Ingenol-3-acylates i

ABSTRACT

The invention relates to compounds of general formula (I) wherein R is (C 1 -C 7 )alkyl, (C 2 -C 7 )alkenyl or (C 2 -C 7 )alkynyl; wherein R is substituted with R1; and pharmaceutically acceptable salts, hydrates, or solvates thereof, for use—alone or in combination with one or more other pharmaceutically active compounds—in therapy, for preventing, treating or ameliorating diseases or conditions responsive to stimulation of neutrophil oxidative burst, responsive to stimulation of keratinocyte IL-8 release or responsive to induction of necrosis.

FIELD OF THE INVENTION

This invention relates to novel derivatives of 3-acyl-ingenol andderivatives thereof and their use as a medicament and in therapy. Theinvention also provides pharmaceutical compositions comprising saidcompounds and methods of treating diseases with said compounds.

BACKGROUND OF THE INVENTION

Ingenol-3-angelate (PEP005, ingenol mebutate) is a diterpene-ester ofthe ingenol family which is isolated from various Euphorbia species,particularly from Euphorbia peplus. The compound is presently subjectfor clinical development for the treatment of actinic keratosis and fornon-melanoma skin cancer.

A number of other ingenol-3-acylates, mainly of long-chain saturated andunsaturated aliphatic fatty acids, have been isolated from variousEuphorbia species [H. Gotta, Z. Naturforschung, (1984), 39b, 683-94; K.Abo, Fitoterapia, (1988), 244-46, S. Zayed, J. Cancer Res. Clin. Oncol.(2001), 127, 40-47]. Furthermore, a small number ingenol-3-acylates havebeen prepared by semi-synthesis (B. Sorg et. al., Z. Naturforsch.,(1982), 37b, 748-56). Some of these ingenol derivatives have beendescribed and tested to be strong irritants and strong tumor-promotingagents. [B. Sorg et. al., Z. Naturforsch., (1982), 37b, 748-56; B. Sorget. al., Carcinogenesis, (1987), 8, 1-4].

Besides the aliphatic ingenol esters also aromatic esters of ingenol areknown. Milliamine C, an ingenol-3-anthraniloate derivative was described(Marston, A. Planta Medica, (1983), 47, 141-47). Also ingenol-3-benzoatehas been described (Sorg, B.; Z Naturforschung, (1982), 37b, 748-56).

Angelic acid and angelic acid esters, as present in ingenol-3-angelate,are prone to isomerisation of the double bond to form the tiglate ester,particularly at basic pH [Beeby, P., Tetrahedron Lett. (1977), 38,3379-3382, Hoskins, W. M., J. Chem. Soc. Perkin Trans. 1, (1977),538-544, Bohlmann, F. et. al., Chem. Ber. (1970), 103, 561-563].

Furthermore, ingenol-3-acylates are known to be unstable as theyrearrange to afford the ingenol-5-acylates and ingenol-20-acylates[Sorg, B. et. al, Z. Naturforsch., (1982), 37B, 748-756].

WO99/08994 describes isolation of compounds from Euphorbia plant andtheir use in cancer and other neoplastic diseases hereunder actinickeratosis or solar keratosis. WO01/93883 describes ingenol derivativesdifferent from the present invention for prophylaxis of a PKC-relatedcondition or disorder in a subject. Diseases mentioned in WO01/93883are: asthma, atherosclerosis, atopic dermatitis, autoimmune disease,bipolar disorder, blood disorder, cardiac hypertrophy, depression,diabetes, hypertension, hyperplastic dermatosis, multiple sclerosis,myocardial ischemia, osteoarthritis, psoriasis, rheumatoid arthritis,transplantation and latent virus. WO01/93884 discloses ingenolderivatives different from the present invention, and their use intreating inflammatory conditions such as resulting from pathogenicorganisms, virus, yeast, fungus, worms, insects, arachnids, nematodes,aemobe etc. WO01/93885 describes ingenol derivatives different from thepresent invention for immunopotentiation. WO08/131491 describes ingenolderivatives different from the present invention for HPV virusinfections. WO06/063382 discloses ingenol derivatives different from thepresent invention for treatment of solid cancers. AU 2006201661discloses a method for treating acute myeloid leukemia usingingenol-3-angelate. WO02/11743 describes a particular use in prostateand bladder cancer. Ingenol derivatives are described in WO07/059584 forpromoting wound healing. WO2010/091472 describes use of ingenols andderivatives in other cosmetic applications.

Ingenol-3-angelate is believed to have a dual mode of action: 1)Induction of cell death by direct cytoxicity or induction of apoptosisand 2) an immunostimulatory effect dominated by neutrophil recruitmentand activation (Rosen, R. H., et al., J Am Acad Derm (2011), e-publishedNovember 2011; Ersvaer, E., et al., Toxins, (2010), 2, 174-194).Nanomolar concentrations of the agent cause activation and modulation ofprotein kinase C (PKC) classical and novel isoforms, with particularimportance of PKCdelta. Through activation of PKCdelta the agent inducesapoptosis in susceptible cells (Hampson, P., et al., Blood, (2005), 106,1362-1368; Cozzi, S. J.,et al., Cancer Res, (2006), 66, 10083-10091).Rapid cytotoxicity on cancer cells is observed at high micromolarconcentrations (Ogbourne, S. M., et al., Cancer Res (2004), 64,2833-2839).

Through activation of various PKC isoforms the agent also inducespro-inflammatory effects, including release of pro-inflammatorymediators (Challacombe, J. M., et al., J Immunol (2006), 177, 8123-8132,activation of vascular endothelium (Hampson, P., et al., Cancer ImmunolImmunother, (2008), 57, 1241-1251); chemoattraction of neutrophilsthrough induction of interleukin 8 in keratinocytes and development ofspecific anti-cancer immune responses by CD8+ cells through adjuvantproperties in animal models (Le, T. T., et al., Vacccine, (2009), 27,3053-3062).

Compounds exerting dual mode of action by induction of cell death bydirect cytoxicity or induction of apoptosis, and by an immunostimulatoryeffect involving neutrophil recruitment and activation, may be usefulfor treatment of conditions associated with hyperplasia or neoplasia.Compounds inducing cell death by primary and/or secondary necrosis andcompounds exhibiting a pro-apoptotic effect may reduce unwanted cellgrowth and remove unwanted cells, and furthermore, stimulation of theinnate immune response and adjuvant effects may augment the biologicalresponse against aberrant or transformed cells.

Compounds inducing cell death by primary and/or secondary necrosis maybe useful for treatment of cosmetic conditions, as these compounds maykill or remove unwanted tissue or cells.

There is a need to find new ingenol derivatives, with a similar orimproved biological activity compared to ingenol-3-angelate.Furthermore, there is a need to find new ingenol derivatives whichinduce cell death by cytotoxicity or apoptosis and/or induce animmunostimulatory effect

The present invention provides branched or substituted 3-O-acyl ingenolderivatives useful for treatment of conditions associated with the useof ingenol-3-angelate or useful for conditions which are affected byinduction of cell death by cytoxicity or induction of apoptosis and/orby an immunostimulatory effect.

Compounds of the present invention stimulate neutrophil oxidative burst,which is part of the innate immune response.

Compounds of the present invention stimulate keratinocyte IL-8 release,thus inducing an immunostimulatory effect.

Some compounds of the present invention induce rapid necrosis.

Compounds of the present invention exhibit suitable stability.

Some compounds of the present invention exhibit improved activity inneutrophil oxidative burst assay compared to to ingenol-3-angelate.

Some compounds of the present invention exhibit improved activity inIL-8 release assay compared to to ingenol-3-angelate.

Some compounds of the present invention exhibit improved activity innecrosis assay compared to ingenol-3-angelate.

SUMMARY OF THE INVENTION

In an embodiment the invention provides a compound of the generalformula I

-   wherein R is (C₁-C₇)alkyl, (C₂-C₇)alkenyl or (C₂-C₇)alkynyl; wherein    R is substituted one or more times with substituents independently    selected from R1; wherein-   R1 represents-   (a) Fluoro, cyano or hydroxy; or-   (b) (C₁-C₄)alkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl,    heterocycloalkyl, heterocycloalkenyl or (C₃-C₆)cycloalkylidene each    of which may optionally be substituted by one or more substituents    independently selected from R2, wherein R2 represents halogen,    cyano, hydroxy, (C₁-C₄)alkyl, (C₂-C₄)alkenyl, halo(C₁-C₄)alkyl,    —NRaCORb, —COORc, —OCORa, —CONRaRb, —OCONRaRb, —NRaCOORb,    —NRaCONRaRb, —NRaSO2Rb, —NRaSO2NRaRb, —SO2NRaRb, —SO2Ra, —S(O)Ra,    —ORa, —SRa, ═N—ORa, ═O; or-   (c) —NRaCORb, —COORc, —OCORa, —CONRaRb, —OCONRaRb, —NRaCOORb,    —NRaCONRaRb, —NRaSO2Rb, —NRaSO2NRaRb, —SO2NRaRb, —SO2Ra, —S(O)Ra,    —ORa, —SRa, ═N—ORa, —NRaRb or ═O;-   and wherein-   Ra and Rb independently represents hydrogen, (C₁-C₄)alkyl,    halo(C₁-C₄)alkyl, (C₁-C₄)alkoxyl(C₁-C₄)alkyl, hydroxy(C₁-C₄)alkyl,    cyano(C₁-C₄)alkyl or aryl;-   and wherein-   Rc represents (C₁-C₄)alkyl, halo(C₁-C₄)alkyl,    (C₁-C₄)alkoxyl(C₁-C₄)alkyl, hydroxy(C₁-C₄)alkyl or    cyano(C₁-C₄)alkyl,-   and pharmaceutically acceptable salts, prodrugs, hydrates and    solvates thereof;-   with the proviso when R1 represents alkyl or alkenyl the combined    length of R and R1 does not exceed that of a chain of 7 carbon    atoms,-   and with the proviso that R and R1 in combination may not be a    straight unbranched or unsubstituted alkyl chain or a straight    unbranched or unsubstituted alkenyl chain,-   and with the proviso that R, or R and R1 in combination is not    trans-2-buten-2-yl.

In an embodiment the invention provides a compound of formula I, for useas a medicament in therapy.

In an embodiment the invention provides a pharmaceutical compositioncomprising a compound of formula I or a pharmaceutically acceptablestereoisomer, salt or in vivo hydrolysable ester thereof together with apharmaceutically acceptable vehicle or excipient.

In an embodiment the invention provides a pharmaceutical compositionsuitable for topical administration comprising a compound of formula Ior a pharmaceutically acceptable stereoisomer, salt or in vivohydrolysable ester thereof together with a pharmaceutically acceptablevehicle or excipient.

In an embodiment the invention provides a compound of formula I for usein the treatment, prevention, amelioration or prophylaxis ofphysiological disorders or diseases associated with hyperplasia orneoplasia.

In an embodiment the invention provides use of a compound of formula Ifor the manufacture of a medicament for the treatment, amelioration orprophylaxis of physiological disorders or diseases associated withhyperplasia or neoplasia.

In an embodiment the invention provides a method of preventing,treating, amelioration or prophylaxis of physiological disorders ordiseases associated with hyperplasia or neoplasia by administration to asubject in need thereof a compound of formula I.

In an embodiment the invention provides a compound of formula I for usein the treatment or amelioration of cosmetic indications.

In an embodiment the invention provides use of compound according toformula I for the manufacture of a medicament for the treatment oramelioration of cosmetic indications.

In an embodiment the invention provides a method of treatment oramelioration of cosmetic indications by administration to a subject inneed thereof a compound of formula I.

In an embodiment the invention provides a pharmaceutical compositioncomprising a compound of formula I or a pharmaceutically acceptablestereoisomer, salt or in vivo hydrolysable ester thereof in combinationwith one or more other therapeutically active agents.

DETAILED DESCRIPTION OF THE INVENTION

In an embodiment the invention provides a compound of the generalformula I above, wherein R is a (C₁-C₇)alkyl, (C₂-C₇)alkenyl or(C₂-C₇)alkynyl; wherein R is substituted one or more times withsubstituents independently selected from R1 wherein

-   R1 represents-   (a) Fluoro, cyano or hydroxy; or-   (b) (C₁-C₄)alkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl,    heterocycloalkyl, heterocycloalkenyl, each of which may optionally    be substituted by one or more substituents independently selected    from R2, wherein R2 represents halogen, cyano, hydroxy,    (C₁-C₄)alkyl, (C₂-C₄)alkenyl, halo(C₁-C₄)alkyl, —NRaCORb, —COORc,    —OCORa, —CONRaRb, —OCONRaRb, —NRaCOORb, —NRaCONRaRb, —NRaSO2Rb,    —NRaSO2NRaRb, —SO2NRaRb, —SO2Ra, —S(O)Ra, —ORa, —SRa, ═N—ORa, ═O; or-   (c) —NRaCORb, —COORc, —OCORa, —CONRaRb, —OCONRaRb, —NRaCOORb,    —NRaCONRaRb, —NRaSO2Rb, —NRaSO2NRaRb, —SO2NRaRb, —SO2Ra, —S(O)Ra,    —ORa, —SRa, ═N—ORa-   and wherein-   Ra and Rb represents hydrogen, (C₁-C₄)alkyl, halo(C₁-C₄)alkyl,    (C₁-C₄)alkoxyl(C₁-C₄)alkyl, hydroxy(C₁-C₄)alkyl, cyano(C₁-C₄)alkyl,-   and wherein-   Rc represents (C₁-C₄)alkyl, halo(C₁-C₄)alkyl,    (C₁-C₄)alkoxyl(C₁-C₄)alkyl, hydroxy(C₁-C₄)alkyl, cyano(C₁-C₄)alkyl-   with the proviso when R1 represents alkyl or alkenyl the combined    length of R and R1 does not exceed that of a chain of 7 carbon    atoms,-   and with the proviso that R and R1 in combination may not be a    straight unbranched or unsubstituted alkyl or alkenyl chain,-   with the proviso that R, or R and R1 in combination is not    trans-2-buten-2-yl;

In an embodiment the invention provides a compound of formula I whereinR1 is located in alpha- or beta-position relative to the carbonyl-group.

In an embodiment the invention provides a compound of formula I whereinR is substituted by R1 in the alpha- or beta-position relative to thecarbonyl-group.

In an embodiment the invention provides a compound of formula I whereinR is substituted by R1 in the alpha-position relative to thecarbonyl-group.

In an embodiment the invention provides a compound of formula I whereinR is (C₁-C₇)-alkyl.

In an embodiment the invention provides a compound of formula I whereinR is ethyl, propyl, tert-butyl, isopropyl, 2-butyl or 3-pentyl.

In an embodiment the invention provides a compound of formula I whereinR is ethyl, propyl, tert-butyl, isopropyl, 2-butyl,3-pentyl or isobutyl.

In an embodiment the invention provides a compound of formula I whereinR is (C₂-C₇)-alkenyl.

In an embodiment the invention provides a compound of formula I whereinR is propenyl, ethenyl.

In an embodiment the invention provides a compound of formula I whereinR is propenyl or ethenyl.

In an embodiment the invention provides a compound of formula I whereinR1 is (C₁-C₄)-alkyl.

In an embodiment the invention provides a compound of formula I whereinR1 is C₁-C₄-alkyl, aryl, cycloalkyl, cycloalkylidene, ═N—ORa, ═O,—NRaRb, —COORc or cyano.

In an embodiment the invention provides a compound of formula I whereinR1 is ethyl or methyl.

In an embodiment the invention provides a compound of formula I whereinR1 is ethyl, methyl, phenyl, cyclohexyl, ═N—OCH3, —N(CH₃)(C₆H₅), —COOCH₃or cyclohexylidene.

In an embodiment the invention provides a compound of formula I whereinR1 is methyl.

In an embodiment the invention provides a compound of formula I whereinR2 is methyl.

In an embodiment the invention provides a compound of formula I saidcompound being:

-   Ingenol 3-(2-methyl-acrylate),-   Ingenol 3-(3-methyl-butenoate),-   Ingenol 3-(2,3-dimethyl-butenoate),-   Ingenol 3-(2-methylene-butyrate),-   Ingenol 3-(2-methyl-propanoate),-   Ingenol 3-(3-methyl-butanoate),-   Ingenol 3-(2RS-methyl-butanoate),-   Ingenol 3-(3,3-dimethyl-butanoate),-   Ingenol 3-(2-ethyl-butanoate),-   Ingenol 3-(2R-methyl-butanoate),-   Ingenol 3-tiglate,-   Ingenol 3-(phenyl-acetate),-   Ingenol 3-(2Z-(methoxycarbonyl)-acrylate),-   Ingenol 3-(2-cyclohexylpropanoate),-   Ingenol 3-((2Z)-2-methoxyimino-2-phenyl-acetate),

Ingenol 3-((2E)-2-methoxyimino-2-phenyl-acetate),

-   Ingenol 3-((Z)-2-methyl-3-(p-tolyl)prop-2-enoate),-   Ingenol 3-((E)-2-phenylbut-2-enoate),-   Ingenol 3-(2,2-diphenylacetate),-   Ingenol 3-(2-cyano-2-cyclohexylidene-acetate) or-   Ingenol 3-(2-(methyl(phenyl)amino)-2-oxo-acetate).

In an embodiment the invention provides a compound of formula I, saidcompound being Ingenol 3-(2-methylene-butyrate).

In an embodiment the invention provides a compound of formula I, saidcompound being Ingenol 3-((Z)-2-methyl-3-(p-tolyl)prop-2-enoate).

In an embodiment the invention provides a compound of formula I, saidcompound being Ingenol 3-(2,3-dimethyl-butenoate).

In an embodiment the invention provides a compound of formula I, saidcompound being Ingenol 3-(2-ethyl-butanoate).

In an embodiment the invention provides a compound of formula I, saidcompound being Ingenol 3-(2-cyclohexylpropanoate).

In an embodiment the invention provides a compound of formula I, saidcompound being Ingenol 3-((E)-2-phenylbut-2-enoate).

Definitions

In the present context, the term “(C_(a)-C_(b))alkyl” wherein a and bare integers refers to a straight or branched chain alkyl radical havingfrom a to b carbon atoms. Thus when a is 1 and b is 7, for example, theterm includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, isohexyl and heptyl.

The term “carbocyclic” refers to a mono-, bi- or tricyclic radicalhaving up to 13 ring atoms, all of which are carbon, and includes aryl,cycloalkyl and cycloalkenyl.

The term “cycloalkyl” refers to a mono-,bi- or tricyclic saturatedcycloalkane radical, comprising 3-13 carbon atoms and includes, forexample, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, bicyclo[2.2.1]heptanyl and adamantyl.

The term “(C_(a)-C_(b))alkenyl” wherein a and b are integers refers to amono-, di- or tri-unsaturated straight or branched chain alkenyl radicalhaving from a to b carbon atoms. Thus when a is 1 and b is 7, forexample, the term includes ethenyl, allyl, propenyl; 1-, 2- or3-butenyl; 1-, 2-, 3- or 4-pentenyl; 1-, 2-, 3-, 4- or 5-hexenyl.

The term “cycloalkenyl” refers to mono-, di- or triunsaturatednon-aromatic cyclic hydrocarbons radicals, including polycyclicradicals, comprising 3-13 carbon atoms and includes, for example,cyclopropenyl, cyclobutenyl, cyclopentenyl or cyclohexenyl.

The term “(C_(a)-C_(b))alkynyl” wherein a and b are integers refers to astraight or branched chain hydrocarbon radical having from a to b carbonatoms comprising 1-2 C—C triple bonds. Thus when a is 1 and b is 7, forexample, the term includes ethynyl, propynyl, butynyl, pentynyl orhexynyl.

The term “heterocyclic” refers to a carbocyclic radical as definedabove, comprising 1-4 heteroatoms, selected from O, N, or S, andincludes heteroaryl, heterocycloalkyl and heterocycloalkenyl.

The term “heterocycloalkyl” refers to a cycloalkyl radical as definedabove, including polycyclic radicals, optionally fused with carbocyclicrings, comprising 1-4 heteroatoms, selected from O, N, or S, e.g.tetrahydrofuranyl, pyrrolidinyl, dioxolanyl, morpholinyl,imidazolidinyl, piperidinyl or 5-oxabicyclo[2.2.2]octane.

The term “heterocycloalkenyl” refers to a cycloalkenyl radical asdefined above, including polycyclic radicals, optionally fused withcarbocyclic rings, comprising 1-4 heteroatoms, selected from O, N, or S,e.g. dihydropyranyl.

The term “aryl” refers to a radical of aromatic carbocyclic ringscomprising 6-10 carbon atoms, in particular phenyl, and optionally fusedcarbocyclic rings with at least one aromatic ring. Thus the termincludes for example phenyl, naphthyl, indenyl or indanyl.

The term “heteroaryl” refers to radicals of heterocyclic aromatic rings,optionally fused with carbocyclic rings or heterocyclic rings,comprising 1-4 heteroatoms, selected from O, S and N, and 1-12 carbonatoms, in particular 5- or 6-membered rings with 1-4 heteroatoms, oroptionally fused bicyclic rings with 1-4 heteroatoms, and wherein atleast one ring is aromatic. Thus the term includes, for example,pyridyl, quinolyl, isoquinolyl, indolyl, tetrazolyl, furyl, thiazolyl,imidazolyl, imidazo[1,2-a]pyrimidinyl, pyrrolyl, pyrazolyl, oxazolyl,isoxazolyl, oxadiazolyl, 1,2,4-triazolyl, thienyl, pyrazinyl,pyrimidinyl, 1,2,3-triazolyl, isothiazolyl, imidazo[2,1-b]thiazolyl,benzimidazolyl, benzofuranyl, benzofuranyl, benzothiophenyl,benzothiazolyl, benzooxazolyl, indazolyl.

The term “halogen” is intended to indicate a substituent from the 7thmain group of the periodic table, preferably fluoro, chloro and bromo.

The term “alkoxy” is intended to indicate a radical of the formula —OR,wherein R is alkyl as indicated above, e.g. methoxy, ethoxy, n-propoxy,isopropoxy, butoxy, etc.

The term hydroxyalkyl is intended to indicate a primary, secondary ortertiary radical of the formula —R—OH, wherein R is alkyl as indicatedabove, e.g. hydroxymethyl or hydroxyethyl.

The term cyanoalkyl is intended to indicate a primary, secondary ortertiary radical of the formula —R—CN, wherein R is alkyl as indicatedabove, e.g. cyanomethyl or cyanoethyl.

The term haloalkyl is intended to indicate a primary, secondary ortertiary radical of the formula —R—X₍₁₋₃₎, wherein R is alkyl asindicated above, and X is halogen as indicated above, e.g.trifluoromethyl, 2,2,2-trifluoroethyl or difluoromethyl.

The term “alkoxyalkyl” is intended to indicate an alkyl radical asdefined above, which is substituted with an alkoxy radical as definedabove, i.e. —R—O—R, wherein each R is alkyl, same or different, asindicated above, e.g. methoxymethyl, ethoxymethyl.

In the present context, the term “cycloalkylidene” is intended toindicate a bivalent radical of cycloalkyl, such as cyclohexylidene,cyclopentylidene or cyclobutylidene as indicated below

In the present context, the term “trans-2-buten-2-yl” is intended toindicate the radical of the structure indicated below

In the present context, a substituted alkyl, a substituted alkenyl, or asubstituted alkynyl is intended to mean an alkyl, an alkenyl, or analkynyl moiety which is substituted with one or more substituent groupsattached to the alkyl, alkenyl, or alkynyl moiety.

The term ‘substituted’ as applied to any moiety herein is intended toindicate substitution with compatible substituents.

In the present context, a straight unbranched alkyl chain is intended toindicate an unbranched alkyl moiety, such as ethyl, n-propyl, n-butyl,n-pentyl, n-hexyl or n-heptyl.

In the present context, a straight unbranched alkenyl chain is intendedto indicate an unbranched alkenyl moiety, such as ethenyl, allyl,propen-1-yl, buten-1-yl, penten-1, hexen-1-yl or hepten-1-yl.

In the present context, an unsubstituted alkyl chain is intended toindicate a straight alkyl moiety with no substituents attached to thealkyl moiety, such as ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl orn-heptyl.

In the present context, an unsubstituted alkenyl chain is intended toindicate a straight alkenyl moiety with no substituents attached to thealkenyl moiety, such as ethenyl, allyl, propen-1-yl, buten-1-yl,penten-1-yl, hexen-1-yl or hepten-1-yl.

In the present context, a substituent (R1) in the alpha-positionrelative to the carbonyl group is intended to indicate a substituent(R1) alpha to the carbonyl moiety as indicated below

In the present context, a substituent (R1) in the beta-position relativeto the carbonyl group is intended to indicate a substituent (R1) beta tothe carbonyl moiety as indicated below

The term “pharmaceutically acceptable salt” is intended to indicatesalts prepared by reacting a compound of formula I comprising a basicmoiety with a suitable inorganic or organic acid, such as hydrochloric,hydrobromic, hydroiodic, sulfuric, nitric, phosphoric, formic, acetic,2,2-dichloroacetic, choline, adipic, ascorbic, L-aspartic, L-glutamic,galactaric, lactic, maleic, L-malic, phthalic, citric, propionic,benzoic, glutaric, gluconic, D-glucuronic, methanesulfonic, salicylic,succinic, malonic, tartaric, benzenesulfonic, ethane-1,2-disulfonic,2-hydroxy ethanesulfonic acid, toluenesulfonic, sulfamic or fumaricacid. Pharmaceutically acceptable salts of compounds of formula Icomprising an acidic moiety may also be prepared by reaction with asuitable base such as sodium hydroxide, potassium hydroxide, magnesiumhydroxide, calcium hydroxide, ammonia, or suitable non-toxic amines,such as lower alkylamines, for example triethylamine, hydroxy-loweralkylamines, for example 2-hydroxyethylamine,bis-(2-hydroxyethyl)-amine, cycloalkylamines, for exampledicyclohexylamine, or benzylamines, for exampleN,N′-dibenzylethylenediamine, and dibenzylamine, or L-arginine orL-lysine.

The present invention further includes prodrugs of compounds of generalformula I, such as esters, acetals, ketals, or other derivatives whichundergo a biotransformation in vivo before exhibiting theirpharmacological effects.

The term “solvate” is intended to indicate a species formed byinteraction between a compound, e.g. a compound of formula I, and asolvent, e.g. alcohol, glycerol or water, wherein said species are in asolid form. When water is the solvent, said species is referred to as ahydrate.

The compounds of formula I may be obtained in crystalline form eitherdirectly by concentration from an organic solvent or by crystallisationor recrystallisation from an organic solvent or mixture of said solventand a cosolvent that may be organic or inorganic, such as water. Thecrystals may be isolated in essentially solvent-free form or as asolvate, such as a hydrate. The invention covers all crystallinemodifications and forms and also mixtures thereof.

The term “cancer” in the context of the present invention is intended tocover skin cancer such as non-melanoma skin cancer, malignant melanoma,Merkel cell carcinoma, squamous cell carcinoma, basal cell carcinoma.Basal cell carcinomas covers as well superficial basal cell carcinomasas nodular basal cell carcinoma. Squamous cell carcinoma covers squamouscell carcinoma in situ (Bowen's disease), invasive squamous cellcarcinoma, cutaneous squamous cell carcinoma, mucosal squamous cellcarcinoma, head and neck squamous cell carcinoma. Other cancer typesincludes haematological cancer such as myeloid cancers in particularsuch as acute myeloid leukemia and chronic myeloid leukemia; Cancer ofthe prostate and bladder including benign prostatic hyperplasia,prostatis intraepithelial carcinoma, carcinoma of the bladder,adenocarcinoma of the prostate and renal cell carcinoma. Other cancerinclude AIDS related cancer, acoustic neoma, adenocystic carcinoma,adrenocortical cancer, agnogenic myeloid metaplasia, alopecia, alveolarsoft-part sarcoma, anal cancer, angiosarcoma, aplastic anaemia,astrocytoma, ataxia-telangiectasia, basal cell carcinoma (bcc), bladdercancer, bone cancers, bowel cancer, brain stem glioma, brain and CNScancers, breast cancer, CNS cancers, carcinoid cancers, cervical cancer,childhood brain cancers, childhood cancer, childhood soft tissuesarcoma, chondrosarcoma, choriocarcinoma, colorectal cancers, cutaneousT-CeIl lymphoma, dermatof[iota]brosarcoma-protuberans, desmoplasticsmall round cell cancer, ductal carcinoma, endocrine cancers,endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma,extra hepatic bile duct cancer, eye cancer, eye: melanoma,retinoblastoma, fallopian tube cancer, fanconi anaemia, fibrosarcoma,gall bladder cancer, gastric cancer, gastrointestinal cancers,gastrointestinal carcinoid cancer, genitourinary cancers, germ cellcancers, gestational trophoblastic disease, glioma, gynecologicalcancers, hematological malignancies, including acute myeloid leukemia,and neck cancer, hepatocellular cancer, hereditary breast cancer,histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiformmole, hypercalcemia, hypopharynx cancer, intra-ocular melanoma, isleT-cell cancer, Kaposi's sarcoma, kidney cancer, Langerhan's cellhistiocytosis, laryngeal cancer, leiomyosarcoma, li-fraumeni syndrome,lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema,lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, male breastcancer, malignant rhabdoid cancer of kidney, medulloblastoma,mesothelioma, metastatic cancer, mouth cancer, multiple endocrineneoplasia, mycosis fungoides, myelodysplastic syndromes, myeloma,myeloproliferative disorders, nasal cancer, nasopharyngeal cancer,nephroblastoma, neuroblastoma, neurofibromatosis, nijmegen breakagesyndrome, non-small cell lung cancer (nscic), ocular cancers,oesophageal cancer, oral cavity cancer, oropharynx cancer, osteosarcoma,ostomy ovarian cancer, pancreas cancer, paranasal cancer, parathyroidcancer, parotid gland cancer, penile cancer, peripheral neuroectodermalcancers, pituitary cancer, polycythemia vera, prostate cancer, rarecancers and associated disorders, retinoblastoma, rhabdomyosarcoma,rothmund Thomson syndrome, salivary gland cancer, sarcoma, schwannoma,sezary syndrome, small cell lung cancer (scic), small intestine cancer,soft tissue sarcoma, spinal cord cancers, stomach cancer, synovialsarcoma, testicular cancer, thymus cancer, thyroid cancer, transitionalcell cancer (bladder), transitional cell cancer (renal-pelvis−/−ureter),trophoblastic cancer, urethral cancer, urinary system cancer,uroplakins, uterine sarcoma, uterus cancer, vaginal Cancer, vulvacancer, Waldenstrom's macroglobulinemia and Wilms' Cancer. The solidcancer which is treated using the methods of the present invention maybe a primary lesion or may be the result of metastasis of a primarycancer. Furthermore, if the solid cancer is a metastasis of a primarycancer, the primary cancer may be either a primary solid cancer asdescribed above or may be a dispersed primary cancer.

In an embodiment of the invention “cancer” is skin cancer. Inembodiments of the invention, skin cancer is non-melanoma skin cancer,malignant melanoma, Merkel cell carcinoma, squamous cell carcinoma,squamous cell carcinoma, basal cell carcinoma such as superficial basalcell carcinomas or nodular basal cell carcinoma.

The term “photodamaged skin” in the context of the present invention isintended to cover fine lines, wrinkles and UV-ageing. UV ageing is oftenmanifested by an increase in the epidermal thickness or epidermalatrophy and most notably by solar elastosis, the accumulation of elastincontaining material just below the dermal-epidermal junction. Collagenand elastic fibres become fragmented and disorganised. At a cosmeticlevel this can be observed as a reddening and/or thickening of the skinresulting a a lethery appearance, skin fragility and irregularpigmentation, loss of tone and elasticity, as well as wrinkling,dryness, sunspots and deep furrow formation.

The term “viral infections” in the context of the present invention isintended to cover HPV infections leading to formation of warts on thebody, such as the skin, genitals and mouth. HPV refers to humanpapilloma virus. Other viruses are selected from adeno-, papova-,herpes- (such as simplex) varicella-zoster, Epstein-Barr-, CMV-, Pox-(such as small pox-) vaccinia-, hepatitis A-, hepatitis B-, hepatitisC-, Rhino-, polio-,rubella-, arbo-, rabies-, influenza-A and B,measles-, mumps-viruses, and HIV, HTLV I and II. In an embodiment of theinvention HPV infection refers to common warts or genital warts.

The term “bacterial infections” in the context of the present inventionis intended to cover prokaryotic and eukaryotic bacterial infections andGram positive and Gram negative and Gram variable bacteria andintracellular bacteria. Examples of bacteries includes Treponema,Borrelia, Neisseria, Legionella, Bordetella, Escherichia, Salmonella,Shigella, Klebsiella, Yersinia, Vibrio, Hemophilus, Rickettsia,Chlamydia, Mycoplasma, Staphylococcus, Streptococcus, Bacillus,Clostridium, Corynebacterium, Proprionibacterium, Mycobacterium,Ureaplasma and Listeria. In particular the species: Treponema pallidum,Borrelia Burgdorferi, Neisseria gonorrhoea, Legionella pneumophila,Bordetella pertussis, Escherichia coli, Salmonella typhi, salmonellatyphimurium, Shigella dysenteriae, Klebsiella pneumoniae, Yersiniapestis, Vibrio cholerae, Hemophilus influenza, Rickettsia rickettsii,Chlamydia trachomatis, Mycoplasma pneumonia, Staphylococcus aureus,Streptococcus pneumoniae, Streptococcus pyogenes, Bacillus anthracis,Clostridium botulinum, Clostridium tetani, clostridium perfringens,Corynebacterium diphteriae, Proprionibacterium acne, Mycobacteriumtuberculosis, Mycobacterium leprae and Listeriare monocytogenes. Lowereukaryotic organism includes yeast and fungus such as Pneumocystisnerinii, Candida albicans, Aspergillus, Histoplasma capsulatum,Blastomyces dermatitidis, Cryptococcus neoformans, Trichophyton andMicrosporum. Complex eukaryotic organism includes worms, insects,aracnids, nematodes, aemobe, Entamoeba histolytica, Giardia lamblia,Trichonomonas vaginalis, Trypanosoma brucei gembiense, Trypanosomacruzi, Blantidium coli, Toxoplasma gondii, Cryptosporidium orLeishmania.

The phrase “physiological disorders or diseases associated withhyperplasia or neoplasia” in the context of the present invention isintended to cover disorders or diseases such as Cutaneous wartsincluding common warts (Verruca vulgaris), plantar warts (Verrucaplantaris) and flat warts (verruca plana); Genital warts (condylomaacuminatum), Pyogenic granuloma, Haemangioma, Scleroderma; Cancers andprecancerous lesions such as Actinic keratosis, Squamous cell carcinomaincluding squamous cell carcinoma in situ (Bowen's disease), invasivesquamous cell carcinoma, cutaneous squamous cell carcinoma, mucosalsquamous cell carcinoma, head and neck squamous cell carcinoma; Basalcell carcinoma including Superficial basal cell carcinoma and Nodularbasal cell carcinoma; Bladder cancer, Lentigo maligna, Cervicaldysplasia, Vulva dysplasia and anal dysplasia, Primary melanoma in situ,Head and neck cancer, Cutaneous metastases of any cancer, Kaposi'ssarcoma, Keratoacanthoma, Merkel cell tumor, Prostate cancer, Mycosisfungoides, Intraepithelial neoplasias including anal, cervical, ductal,oral, perianal, prostatic, penile, vaginal and vulvar intraepithelialneoplasia.

The term “cosmetic indications” in the context of the present inventionis intended to cover indications such as: Photodamaged skin, Seborrheickeratosis, Scars, Keloids, Melasma, Poikiloderma of Civatte, Tattooremoval, Naevi, Skin tags.

In the context of the present invention the term “wound healing” means:reducing or minimizing scar tissue or improving cosmesis or functionaloutcome in a wound and scar reduction, wherein the wound is cutaneous,chronic or for example diabetes associated, and includes cuts andlacerations, surgical incisions, punctures, graces, scratches,compression wounds, abrasions, friction wounds, chronic wounds, ulcers,thermal effect wounds, chemical wounds, wounds resulting from pathogenicinfections, skin graft/transplant donor and recipient sites, immuneresponse conditions, oral wounds, stomach or intestinal wounds, damagedcartilage or bone, amputation sides and corneal lesions.

The compounds of the present invention are contemplated in the treatmentof cancer, actinic keratosis, seborrheic keratosis, viral infections,bacterial infections, wound healing, and treatment of photodamaged skin.

In an embodiment of the invention the compounds of the invention arecontemplated for use in the treatment of superficial basal cellcarcinoma (BCC), nodular BCC, squamous cell carcinoma or squamous cellcarcinoma in situ (SCCIS).

In an embodiment of the invention the compounds of the invention arecontemplated for use in the treatment of actinic keratosis.

In an embodiment of the invention the compounds of the invention arecontemplated for use in the treatment of Seborrheic keratosis.

In an embodiment of the invention the compounds of the invention arecontemplated for use in the treatment of photodamaged skin.

In an embodiment of the invention the compounds of the invention arecontemplated for use in the treatment of or lesions caused by HPVinfection. In an embodiment of the invention the lesions are commonwarts or genital warts.

In an embodiment of the invention the compounds of the invention arecontemplated for use in the treatment of squamous cell carcinoma in situor invasive squamous cell carcinoma.

In an embodiment of the invention the compounds of the invention arecontemplated for use in the treatment of cutaneous squamous cellcarcinoma, mucosal squamous cell carcinoma or head and neck squamouscell carcinoma.

In an embodiment of the invention the compounds of the invention arecontemplated for use in the treatment of superficial basal cellcarcinoma or nodular basal cell carcinoma.

In an embodiment of the invention the compounds of the invention arecontemplated for use in the treatment of cutaneous warts or genitialwarts.

In an embodiment of the invention the compounds of the invention arecontemplated for use in the treatment of common warts, plantar warts andflat warts.

In an embodiment of the invention the compounds of the invention arecontemplated for use in the treatment of lentigo maligna.

In an embodiment of the invention the compounds of the invention arecontemplated for use in the treatment of cervical intraepithelialneoplasia, anal intraepithelial neoplasia or vulva intraepithelialneoplasia.

In an embodiment of the invention the compounds of the invention arecontemplated for use in the treatment of acute myeloid leukemia.

In an embodiment the invention provides a method of treatment of cancer,actinic keratosis, seborrheic keratosis, viral infections, bacterialinfections, wound healing, and treatment of photodamaged skin byadministration to a subject in need thereof a compound of formula I.

In an embodiment the invention provides a method of treatment actinickeratosis by administration to a subject in need thereof a compound offormula I above.

In an embodiment the invention provides a method of treatment Seborrheickeratosis by administration to a subject in need thereof a compound offormula I above.

In an embodiment the invention provides a method of treatmentphotodamaged skin by administration to a subject in need thereof acompound of formula I above.

In an embodiment the invention provides a method of treatment of lesionscaused by HPV infection by administration to a subject in need thereof acompound of formula I above.

In an embodiment the invention provides a method of treatment of commonwarts or genital warts by administration to a subject in need thereof acompound of formula I above.

In an embodiment the invention provides a method of treatment ofcutaneous squamous cell carcinoma, mucosal squamous cell carcinoma orhead and neck squamous cell carcinoma by administration to a subject inneed thereof a compound of formula I above.

In an embodiment the invention provides a method of treatment of commonwarts, plantar warts and flat warts by administration to a subject inneed thereof a compound of formula I above.

In an embodiment the invention provides a method of treatment of lentigomaligna by administration to a subject in need thereof a compound offormula I above.

In an embodiment the invention provides a method of treatment ofcervical intraepithelial neoplasia, anal intraepithelial neoplasia orvulva intraepithelial neoplasia by administration to a subject in needthereof a compound of formula I above.

In an embodiment the invention provides a method of treatment of acutemyeloid leukemia by administration to a subject in need thereof acompound of formula I above.

In an embodiment the invention provides use a compound according toformula I above in the manufacture of a pharmaceutical composition forthe treatment or amelioration of a disease, disorder or conditionresponsive to stimulation of neutrophil oxidative burst.

In an embodiment the invention provides use of a compound according toformula I above in the manufacture of a pharmaceutical composition forthe treatment or amelioration of a disease, disorder or conditionresponsive to stimulation of keratinocyte IL-8 release.

In an embodiment the invention provides use of a compound according toformula I above in the manufacture of a pharmaceutical composition forthe treatment or amelioration of a disease, disorder or conditionresponsive to induction of necrosis.

In an embodiment the invention provides a method of preventing,treating, amelioration or prophylaxis of physiological disorders ordiseases responsive to stimulation of neutrophil oxidative burst byadministration to a subject in need thereof a compound according toformula I above.

In an embodiment the invention provides a method of preventing,treating, amelioration or prophylaxis of physiological disorders ordiseases responsive to stimulation of keratinocyte IL-8 release byadministration to a subject in need thereof a compound according toformula I above.

In an embodiment the invention provides a method of preventing,treating, amelioration or prophylaxis of physiological disorders ordiseases responsive to responsive to induction of necrosis byadministration to a subject in need thereof a compound according toformula I above.

In an embodiment the invention provides a compound according to formulaI above for use in the treatment or amelioration of a disease, disorderor condition responsive to stimulation of neutrophil oxidative burst.

In an embodiment the invention provides a compound according to formulaI above for use in the treatment or amelioration of a disease, disorderor condition responsive to stimulation of keratinocyte IL-8 release.

In an embodiment the invention provides a compound according to formulaI above for use in the treatment or amelioration of a disease, disorderor condition responsive to induction of necrosis.

In an embodiment the invention provides a method of treatment of acutemyeloid leukemia by administration to a subject in need thereof acompound of formula I above.

In an embodiment the invention provides a compound of formula I, for usein the treatment, prevention, amelioration or prophylaxis ofphysiological disorders or diseases associated with actinic keratosis,seborrheic keratosis, cancer, photodamaged skin or lesions caused by HPVinfection.

In an embodiment the invention provides the use of a compound of formulaI, for the manufacture of a medicament for the treatment, ameliorationor prophylaxis of physiological disorders or diseases associated withactinic keratosis, Seborrheic keratosis, cancer, photodamaged skin orlesions caused by HPV infection.

In an embodiment the invention provides a method of preventing,treating, amelioration or prophylaxis of physiological disorders ordiseases associated with actinic keratosis, Seborrheic keratosis,cancer, photodamaged skin or lesions caused by HPV infection byadministration to a subject in need thereof a compound of formula I.

Pharmaceutical Compositions

For use in therapy, compounds of the present invention are typically inthe form of a pharmaceutical composition. The invention thereforerelates to a pharmaceutical composition comprising a compound of formulaI, together with a pharmaceutically acceptable excipient or vehicle. Theexcipient must be “acceptable” in the sense of being compatible with theother ingredients of the composition and not deleterious to therecipient thereof.

Pharmaceutical compositions of the invention may be in unit dosage formsuch as tablets, pills, capsules, powders, granules, elixirs, syrups,emulsions, ampoules, suppositories or parenteral solutions orsuspensions; for oral, parenteral, opthalmic, transdermal,intra-articular, topical, pulmonal, nasal, buccal or rectaladministration or in any other manner appropriate for the formulation ofcompounds of the invention and in accordance with accepted practicessuch as those disclosed in Remington: The Science and Practice ofPharmacy, 21^(st) ed., 2000, Lippincott Williams & Wilkins.

For oral administration in the form of a tablet or capsule, a compoundof formula I may suitably be combined with an oral, non-toxic,pharmaceutically acceptable carrier such as ethanol, glycerol, water orthe like. Furthermore, suitable binders, lubricants, disintegratingagents, flavouring agents and colourants may be added to the mixture, asappropriate. Suitable binders include, e.g., lactose, glucose, starch,gelatin, acacia gum, tragacanth gum, sodium alginate,carboxymethylcellulose, polyethylene glycol, waxes or the like.Lubricants include, e.g., sodium oleate, sodium stearate, magnesiumstearate, sodium benzoate, sodium acetate, sodium chloride or the like.Disintegrating agents include, e.g., starch, methyl cellulose, agar,bentonite, xanthan gum or the like. Additional excipients for capsulesinclude macrogols or lipids.

For the preparation of solid compositions such as tablets, the activecompound of formula I is mixed with one or more excipients, such as theones described above, and other pharmaceutical diluents such as water tomake a solid preformulation composition containing a homogenous mixtureof a compound of formula I. The term “homogenous” is understood to meanthat the compound of formula I is dispersed evenly throughout thecomposition so that the composition may readily be subdivided intoequally effective unit dosage forms such as tablets or capsules.

In the form of a dosage unit, the compound may be administered one ormore times a day at appropriate intervals, always depending, however, onthe condition of the patient, and in accordance with the prescriptionmade by the medical practitioner. Conveniently, a dosage unit of aformulation contain between 0.01 mg and 200 mg, preferably between 0.01mg and 20 mg, such as 0.01-5 mg of a compound of formula I.

A suitable dosage of the compound of the invention will depend, interalia, on the age and condition of the patient, the severity of thedisease to be treated and other factors well known to the practisingphysician. The compound may be administered either orally, parenterallyor topically according to different dosing schedules, e.g. daily or withweekly intervals. In general a single dose will be in the range from0.01 to 200 mg/kg body weight. The compound may be administered as abolus (i.e. the entire daily dosis is administered at once) or individed doses two or more times a day.

If the treatment involves administration of another therapeuticallyactive compound it is recommended to consult Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9^(th) Ed., J. G. Hardman and L.E. Limbird (Eds.), McGraw-Hill 1995, for useful dosages of saidcompounds. The administration of a compound of the present inventionwith one or more other active compounds may be either concomitantly orsequentially.

Liquid formulations for either oral or parenteral administration of thecompound of the invention include, e.g., aqueous solutions, syrups,aqueous or oil suspensions and emulsion with edible oils such ascottonseed oil, sesame oil, coconut oil or peanut oil. Suitabledispersing or suspending agents for aqueous suspensions includesynthetic or natural gums such as tragacanth, alginate, acacia, dextran,sodium carboxymethylcellulose, gelatin, methylcellulose orpolyvinylpyrolidone.

For parenteral administration, e.g. intramuscular, intraperitoneal,subcutaneous or intravenous injection or infusion, the pharmaceuticalcomposition preferably comprises a compound of formula I dissolved orsolubilised in an appropriate, pharmaceutically acceptable solvent. Forparenteral administration, the composition of the invention may includea sterile aqueous or non-aqueous solvent, in particular water, isotonicsaline, isotonic glucose solution, buffer solution or other solventconventionally used for parenteral administration of therapeuticallyactive substances. The composition may be sterilised by, for instance,filtration through a bacteria-retaining filter, addition of asterilising agent to the composition, irradiation of the composition, orheating the composition. Alternatively, the compound of the inventionmay be provided as a sterile, solid preparation, e.g. a freeze-driedpowder, which is dissolved in sterile solvent immediately prior to use.The composition intended for parenteral administration may additionallycomprise conventional additives such as stabilisers, buffers orpreservatives, e.g. antioxidants such as methyl hydroxybenzoate or thelike.

Compositions for rectal administration may be in the form of asuppository incorporating the active ingredient and a carrier such ascocoa butter, or in the form of an enema. Compositions suitable forintra-articular administration may be in the form of a sterile aqueouspreparation of the active ingredient which may be in microcrystallineform, for example, in the form of an aqueous microcrystallinesuspension. Liposomal formulations or biodegradable polymer systems mayalso be used to present the active ingredient for both intra-articularand ophthalmic administration.

Compositions suitable for topical administration, including ophthalmictreatment, include liquid or semi-liquid preparations such as liniments,lotions, gels, applicants, oil-in-water or water-in-oil emulsions suchas creams, ointments or pastes; or solutions or suspensions such asdrops. Compositions for ophthalmic treatment may preferably additionallycontain a cyclodextrin. Compositions suitable for administration to thenasal or buccal cavity or for inhalation include powder, self-propellingand spray formulations, such as aerosols and atomizers.

Human skin, in particular the outer layer, the stratum corneum, providesan effective barrier against penetration of microbial pathogens andtoxic chemicals. While this property of skin is generally beneficial, itcomplicates the dermal administration of pharmaceuticals in that a largequantity, if not most, of the active ingredient applied on the skin of apatient suffering from a dermal disease may not penetrate into theviable layers of the skin where it exerts its activity.

Penetration of the skin is facilitated by addition of penetrationenhancers which include isopropyl alcohol, sulphoxides, azones,pyrrolidines, alkanols, and glycols. In embodiments of the invention thepenetrations enhancers includes DMSO, laurocapram, 2-pyrrolidone,decanol and propylene glycol. In an embodiment of the invention thepenetration enhancer is isopropyl alcohol.

In embodiments of the invention the therapeutically active compound isdissolved in a suitable solvent. Suitable solvents are glycols, ketone,acetates and ethers. Ingenol compounds have been shown to have goodstability in alcohols such as benzyl alcohol and isopropyl alcohol. Ingeneral, ingenol compounds have previously shown to have good stabilityat low pH. In embodiments of the present invention pH the pharmaceuticalformulation is below 7. In embodiments of the present invention the pHof the pharmaceutical formulation is below 6. In embodiments of thepresent invention the pH of the pharmaceutical formulation is below 4.5.In embodiments of the present invention the pH of the pharmaceuticalformulation is below 4.0. In embodiments of the present invention the pHof the pharmaceutical formulation is below 4.5 and no less than 2.5. Inembodiments of the present invention the pH of the pharmaceuticalformulation is below 4.0 and no less than 2.5. The preferred pH rangecan be obtained by including an appropriate buffer. In an embodiment ofthe invention the buffer is an acetate buffer. In embodiments of theinvention a citrate buffer is used. In embodiments of the invention amixed citrate-phosphate buffer is used.

In one embodiment, the composition is an ointment. According to thecurrent FDA classification, an ointment is a semisolid dosage from whichmay contain water and volatile substances in an amount of up to 20% byweight and which contains more than 50% by weight of hydrocarbons, waxesor polyols in the vehicle. Thus, according to the invention, theointment may be a water-in-oil composition in which case thenanosuspension may be added as such to the lipophilic components of thecomposition, such that the composition contains up to 10% by weight or,preferably, up to 5% by weight of the aqueous phase. Alternatively, thecomposition may be a non-aqueous ointment which contains less than about2%, preferably less than 1%, of free water by weight of the composition.

The ointment carrier may suitably contain a paraffin selected fromparaffins consisting of hydrocarbons with chain lengths from C₅₋₆₀ andmixtures thereof. A frequently used ointment carrier is petrolatum, orwhite soft paraffin, which is composed of hydrocarbons of differentchain lengths, peaking at about C₄₀₋₄₄, or a mixture of petrolatum andliquid paraffin (consisting of hydrocarbons of different chain lengthspeaking at C₂₈₋₄₀). While petrolatum provides occlusion of the treatedskin surface, reducing transdermal loss of water and potentiating thetherapeutic effect of the active ingredient in the composition, it tendsto have a greasy and/or tacky feel which persists for quite some timeafter application, and it is not easily spreadable. It may therefore bepreferred to employ paraffins consisting of hydrocarbons of a somewhatlower chain length, such as paraffins consisting of hydrocarbons withchain lengths peaking at C₁₄₋₁₆, C₁₈₋₂₂, C₂₀₋₂₂, C₂₀₋₂₆ or mixturesthereof. It has been found that such paraffins are more cosmeticallyacceptable in that they are less tacky and/or greasy on application andmore easily spreadable. They are therefore expected to result inimproved patient compliance. Suitable paraffins of this type aremanufactured by Sonneborn and marketed under the trade name Sonnecone,e.g. Sonnecone CM, Sonnecone DM1, Sonnecone DM2 and Sonnecone HV. Theseparaffins are further disclosed and characterized in WO08/141078 whichis incorporated herein by reference. (The hydrocarbon composition of theparaffins has been determined by gas chromatography.)

To impart a desired viscosity to the composition, it may suitablyinclude a lipophilic viscosity-increasing ingredient such as a wax. Thewax may be a mineral wax composed of a mixture of high molecular weighthydrocarbons, e.g. saturated C₃₅₋₇₀ alkanes, such as microcrystallinewax. Alternatively, the wax may be a vegetable or animal wax, e.g.esters of C₁₄₋₃₂ fatty acids and C₁₄₋₃₂ fatty alcohols, such as beeswax.The amount of viscosity-increasing ingredient may vary according to theviscosifying power of the ingredient, but may typically be in the rangeof about 1-20% by weight of the composition. When theviscosity-increasing ingredient is microcrystalline wax it is typicallypresent in an amount in the range of about 5-15% by weight, e.g. about10% by weight, of the composition.

To maintain good physical stability of the composition, in particular toavoid separation of the aqueous and lipid phases therein, it may beadvantageous to include a water-in-oil emulsifier with an HLB value of3-8. Examples of such emulsifiers are polyoxyethylene C₈₋₂₂ alkylethers, e.g. polyoxyethylene stearyl ether, polyoxyethylene cetyl ether,polyoxyethylene oleyl ether or polyoxyethylene lauryl ether. The amountof emulsifier is typically in the range of 2-10% w/w of the composition.In another embodiment, the composition is a cream which may comprisesimilar components to the ointment, but which is typically anoil-in-water-emulsion containing a substantial amount of water.

The composition may also comprise other components commonly used indermal formulations, e.g. antioxidants (e.g. alpha-tocopherol),preservatives such as benzyl alcohol, sodium edetate, pigments, skinsoothing agents, skin healing agents and skin conditioning agents suchas urea, allantoin or bisabolol, cf. CTFA Cosmetic Ingredients Handbook,2^(nd) Ed., 1992. In an embodiment of the invention the preservative isbenzyl alcohol.

In an embodiment the composition is a gel. Suitable gelling agentsinclude, water soluble cellulose derived polymers, such as hydroxyalkylcellulose polymers. In embodiments of the invention the polymers arehydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcelluloseand hydroxypropylmethylcellulose. Other gelling agents are cellulosessuch as carboxymethyl cellulose, methylhydroxyethyl cellulose and methylcellulose, carbomer such as carbopol and carrageenans. In embodiments ofthe invention the gelling agent is cellulose derived. In embodiments ofthe invention the cellulose is a hydroxyalkylcellulose, such ashydroxyethylcellulose.

In an embodiment of the invention the composition comprises activecompound, penetration enhancer, preservative, gelling agent and bufferat a pH of below 4 and not less than 2.5. For topical administration,the compound of formula I may typically be present in an amount of from0.001 to 20% by weight of the composition, such as 0.01% to about 10%.Inembodiments of the present invention the active compound is present in0.05-1%. In an embodiment of the present invention the active compoundis present in 0.01-0.5%. In an embodiment of the present invention theactive compound is present in a concentration of around 0.1%. In anembodiment of the invention the composition comprises 0.005-0,1% activecompound, 20-40% isopropyl alcohol, 0.5-10% benzyl alcohol, 0.5-5%hydroxyl ethyl cellulose and citrate buffer to 100%.

Formulation of ingenol derivatives in a gel for topical application hasbeen described in WO07/068963, which is incorporated herein byreference.

Methods of Preparation

The compounds of formula I may for example be prepared using thereactions and techniques outlined below together with methods known inthe art of synthetic organic chemistry, or variations thereof asappreciated by those skilled in the art. Preferred methods include, butare not limited to, those described below. The reactions are carried outin solvents appropriate to the reagents and materials employed andsuitable for the transformations being effected. Also, in the syntheticmethods described below, it is to be understood that all proposedreaction conditions, including choice of solvent, reaction atmosphere,reaction temperature, duration of experiment and work-up procedures, arechosen to be conditions of standard for that reaction, which should bereadily recognized by one skilled in the art. Not all compounds fallinginto a given class may be compatible with some of the reactionconditions required in some of the methods described. Such restrictionsto the substituents which are compatible with the reaction conditionswill be readily apparent to one skilled in the art and alternativemethods can be used. The compounds of the present invention or anyintermediate may be purified if required using standard methods wellknown to a synthetic organist chemist, e.g. methods described in W.Armarego “Purification of Laboratory Chemicals”, Butterworth-Heinemann,6^(th) ed. 2009. Starting materials are either known compounds,commercially available, or they may be prepared by routine syntheticmethods well known to a person skilled in the art.

The compounds of the invention may for example be prepared according tothe following non-limiting general methods and examples

The compounds of the general formula I can for example be synthesisedaccording to scheme 1 or 2 by reacting ingenol with a hydroxylprotecting agent or a dihydroxyl protecting agent to afford theprotected ingenol derivatives a or c according to methods described in,but not limited to “Protective Groups in Organic Synthesis”, 4th ed. P.G. M. Wuts; T. W. Greene, John Wiley, 2007 or in P. J. Kocienski,“Protecting Groups”, 3rd ed. G. Thieme, 2003 and references citedtherein.

For example compound a, wherein the protective group (Pg) istriphenylmethyl, can be synthesised by reacting ingenol with atriphenylmethyl reagent such as triphenylmethylpyridinium fluoroborateor triphenylmethyl chloride in a suitable solvent such as pyridine,N,N-dimethylformamide or dichloromethane in the presence or in theabsence of base (e.g. Opferkuch et. al., Z. Naturforschung, (1981), 36B,878). Compound a, wherein the protective group (Pg) is silyl, can forexample be synthesised by reacting ingenol with a silyl chloride such astert-butyldimethylsilyl chloride, tert-butyldiphenylsilyl chloride ortriisopropylsilyl chloride in a suitable solvent such asN,N-dimethylformamide, pyridine, dichloromethane, tetrahydrofuran oracetonitrile in the presence of a suitable base such as imidazole,triethylamine, N,N-diisopropylethylamine or4-(N,N-dimethylamino)pyridine (e.g. Sorg, B. et. al, Z. Naturforsch.,(1982), 37B, 1640-47), or by reacting compound (II) with a silyltriflate such as tert-butyldimethylsilyl trifluoromethanesulfonate in asuitable solvent such as dichloromethane in the presence of a suitablebase such as triethylamine.

Compound a wherein Pg is 2-tetrahydropyranyl, can for example besynthesised by reacting ingenol with dihydropyran in a suitable solventsuch as dichloromethane or acetonitrile in the presence of a suitableacid such as p-toluenesulfonic acid.

Compound c wherein the protective group (Pg) represents an acetal suchas benzylidene acetal can for example be prepared by reacting ingenolwith benzaldehyde or benzaldehyde dimethyl acetal in a suitable solventsuch as dichloromethane or N,N-dimethylformamide in the presence of asuitable acid such as p-toluenesulfonic acid.

Compound c wherein the protective group (Pg) represents a ketal such asisopropylidene ketal can for example be synthesised by reacting ingenolwith a ketone such as acetone or a dimethoxy ketal such as 2,2-dimethoxypropane in a suitable solvent such as dichloromethane orN,N-dimethylformamide in the presence of a suitable acid such asp-toluenesulfonic acid (e.g B. Sorg, Z. Naturforsch. (1982), 37b,748-756). Acetone and 2,2-dimethoxy propane can also act as solvents.

As depicted in scheme 1 and 2 the protected ingenol derivatives a or cmay be esterified to give compounds of the general formula b or daccording to methods for esterification of hydroxyl groups described in,but not limited to “Esterification” by J. Otera, Wiley-VCH, 2003 andreferences cited therein. Compound b or d can for example be synthesisedby reacting compound a or c with an activated acid derivative such as anacid halide such as acid chloride. The esterification by reaction withacid chloride can take place in a suitable solvent such asdichloromethane or toluene without an activator, or it can take place inthe presence of a base such as pyridine, triethylamine or4-(N,N-dimethylamino)pyridine (e.g. B. Sorg, Z. Naturforsch. (1982),37b, 748-756).

Compound b or d can for example be synthesised by reacting compound a orc with activated acid derivative such as an acid anhydride. Theesterification by reaction with an acid anhydride can take place withouta catalyst (e.g. Opferkuch et. al., Z. Naturforschung, (1981), 36B,878), or in the presence of an acidic catalyst using an acid such asperchloric acid or a Lewis acid such as scandium (III) triflate orbismuth (III) triflate, or in the presence of a base such as sodiumhydrogencarbonate or triethylamine.

Compound b or d can for example be synthesised by reacting compound a orc with an activated acid derivative such as a mixed anhydride of an acidsuch as trichlorobenzoic acid. The esterification by reaction with amixed anhydride can take place in a suitable solvent without a catalyst,or in the presence of an acidic catalyst using an acid such asperchloric acid or a Lewis acid such as scandium (III) triflate orbismuth (III) triflate, or in the presence of a base such as sodiumhydrogencarbonate or triethylamine.

Compound b or d can for example be synthesised by reacting compound a orc with an acid in the presence a coupling reagent such as a carbodiimidesuch as dicyclohexylcarbodiimide orN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide with or without thepresence of a base such as 4-(N,N-dimethylamino)pyridine and with orwithout catalysts such as 4-(N,N-dimethylamino)pyridine in a suitablesolvent such as dichloromethane (e.g Appendino et. al., Eur. J. Org.Chem. (1999), 3413). Solid-supported coupling reagents can also be usedin the esterification step [Nam, N.-H., Journal of CombinatorialChemistry, (2003), 5, 479-545, or “Esterification” by J. Otera,Wiley-VCH, 2003].

The compounds of formula I may be prepared by selective removal of theprotective groups Pg from the compounds of the general structure b or daccording to methods for deprotection of hydroxyl or dihydroxylprotective groups described, in but not limited to “Protective Groups inOrganic Synthesis”, 4th ed. P. G. M. Wuts; T. W. Greene, John Wiley,2007 or in P. J. Kocienski, “Protecting Groups”, 3rd ed. G. Thieme, 2003and references cited therein.

Compounds of general formula I can for example be prepared fromcompounds of general formula d wherein Pg represents an acetal such asbenzylidene acetal or a ketal such as an isopropyliden ketal by cleavageof the protecting group in the presence of a suitable acid such asaqueous hydrogen chloride, acetic acid, trifluoroacetic acid orp-toluenesulfonic acid in a suitable solvent such as methanol or aqueoustetrahydrofuran.

Compounds of general formula I can for example be prepared fromcompounds of general formula b wherein Pg represents an alkoxyalkyl suchas 2-tetrahydropyranyl by cleaving the acetal moiety, for example byacid catalysed cleavage in the presence of a suitable acid such asp-toluenesulfonic acid in a suitable solvent such as methanol.

Compounds of general formula I can for example be prepared fromcompounds of general formula b wherein Pg represents silyl such astert-butyldimethylsilyl by reacting compound b with a suitable acid suchas hydrogen chloride in a suitable solvent such as methanol or byreacting with a fluoride source such as tetra n-butylammonium fluorideor tetrafluorosilane in a suitable solvent such as tetrahydrofuran oracetonitrile.

Compounds of general formula I can for example be prepared fromcompounds of general formula b wherein Pg represents triphenylmethyl byreacting compound b with a suitable acid such as formic acid ortrifluoroacetic acid in a suitable solvent such as ether, methanol ordichloromethane.

Compounds of formula b, d or I of scheme 1 or 2 above, can for examplebe synthesised enzymatic esterification by reacting compound a, c oringenol with an acyl donor such as an acid anhydride, an ester such asvinyl ester or a thioester in the presence of an enzyme such as a lipaseor an esterase.

EXAMPLES

General All the starting materials used are commercially available,unless otherwise described. For ¹H nuclear magnetic resonance (NMR)spectra, chemical shift values (δ) (in ppm) are quoted;tetramethylsilane (δ=0.00) is as standard. The value of a defineddoublet (d), triplet (t), quartet (q)) or a range (m) is given. Chemicalshifts of exchangeable protons (often broad singlets (bs)) are sometimesdifficult to locate in the spectra. All organic solvents used wereanhydrous, unless otherwise specified. Flash chromatography wasperformed on silica gel. Appropriate mixtures of ethyl acetate andheptane were used as eluents unless otherwise noted. Compounds weredetected on TLC (thin layer chromatography) plates by development withaqueous potassium permanganate solution.

Ingenol-5,20-acetonide

Ingenol (1.00 g, 2.30 mmol) was dissolved in a solution ofp-toluenesulphonic acid monohydrate in acetone (0.47 mg/mL, 22.5 mL).The solution was stirred at room temperature for 25 min. To thissolution was added a saturated aqueous solution of NaHCO₃ (0.2 mL). Theobtained mixture was concentrated in vacuo. The residue was taken up inbrine and extracted with ethyl acetate. The combined organic phases weredried over sodium sulfate and concentrated in vacuo. The residue waspurified by flash chromatography (heptane/ethyl acetate19:1→heptane/ethyl acetate 0:1), giving the title compound as a whitesolid (616 mg, 69%). (See also: Opferkuch, H. J. et.al., Z.Naturforsch., (1981), 86b, 878-887.)

¹H NMR (300 MHz, CDCl₃) δ 5.91 (q, J=1.5 Hz, 1H), 5.79 (m, 1H), 4.25 (d,J=4.5 Hz, 1H), 4.20-4.07 (m, 3H), 3.93 (s, 1H), 3.51 (s, 1H), 2.57-2.41(m, 2H), 2.25 (ddd, J=15.7, 8.4, 2.9 Hz, 1H), 1.85 (d, J=1.5 Hz, 3H),1.77 (dt, J=15.8, 5.9 Hz, 1H), 1.41 (s, 3H), 1.35 (s, 3H), 1.13 (s, 3H),1.05 (s, 3H), 1.00-0.87 (m, 4H), 0.70 (td, J=8.4, 6.4 Hz, 1H).

General Procedures for the Preparation of Compounds of General FormulaII

Procedure a

A mixture of carboxylic acid (0.100 mmol), dicyclohexylcarbodiimide(0.100 mmol), 4-(N,N-dimethylamino)-pyridine (0.0025 mmol) andingenol-5,20-acetonide (0.050 mmol) were stirred at room temperature indichloromethane for 20-24 h. The mixture was mixed with ethyl acetate,filtered and washed with saturated aqueous sodium chloride. The organicphase was dried with sodium sulphate, concentrated in vacuo and purifiedby flash chromatography (heptane→heptane/ethyl acetate 7:3), giving thetitle compound as a white solid.

Procedure b

A mixture of acyl chloride (0.0625 mmol), diisopropylethylamine (0.075mmol), 4-(N,N-dimethylamino)-pyridine (0.070 mmol) andingenol-5,20-acetonide (0.050 mmol) were stirred at 55° C. intetrahydrofuran for 6-20 h. The mixture was mixed with ethyl acetate,filtered and washed with saturated aqueous sodium chloride. The organicphase was dried with sodium sulphate, concentrated in vacuo and purifiedby flash chromatography (heptane→heptane/ethyl acetate 7:3), giving thetitle compound as a white solid.

Procedure c

A mixture of carboxylic acid (0.100 mmol), dicyclohexylcarbodiimide(0.100 mmol), 4-(N,N-dimethylamino)-pyridine (0.025 mmol) andingenol-5,20-acetonide (0.050 mmol) were stirred in a microwave oven at150° C. in acetonitrile for 5 min. The mixture was mixed with ethylacetate, filtered and washed with saturated aqueous sodium chloride. Theorganic phase was dried with sodium sulphate, concentrated in vacuo andpurified by flash chromatography (heptane→heptane/ethyl acetate 7:3),giving the title compound as a white solid.

Procedure d

A mixture of acyl chloride (0.125 mmol), diisopropylethylamine (0.250mmol), 4-(N,N-dimethylamino)-pyridine (0.025 mmol) andingenol-5,20-acetonide (0.050 mmol) were stirred in a microwave oven at150° C. in acetonitrile for 20 min. The mixture was mixed with ethylacetate, filtered and washed with saturated aqueous sodium chloride. Theorganic phase was dried with sodium sulphate, concentrated in vacuo andpurified by flash chromatography (heptane→heptane/ethyl acetate 7:3),giving the title compound as a white solid.

General Procedure for the Preparation of Compounds of General Formula I

Procedure e

Ingenol-5,20-acetonide-3-acylate (0.10 mmol) was dissolved intetrahydrofuran (0.47 mL) under argon. An aqueous solution of HCl (4 M,4.7 μL) was added. The solution was stirred at room temperature for20-27 h. The solution was concentrated in vacuo. The residue waspurified by flash chromatography (heptane/ethyl acetate5:1→heptane/ethyl acetate 3:7), giving the title compound.

Preparation 201:

Ingenol-5,20-acetonide-3-(2-methyl-acrylate) (Compound 201)

Compound 201 was prepared according to Procedure a.

Starting material: 2-Methyl-acrylic acid.

¹H NMR (300 MHz, CDCl₃) δ 6.12-6.11 (m, 1H), 6.05 (m, 1H), 5.79-5-77 (m,1H), 5.62 (s, 1H), 5.60 (m, 1H), 4.25-4.11 (m, 3H), 4.02 (s, 1H), 3.14(s, 1H), 2.61-2.54 (m, 1H), 2.31-2.22 (m, 1H), 1.97 (m, 3H), 1.81-1.72(m, 4H), 1.47 (s, 3H), 1.42 (s, 3H), 1.09 (s, 3H), 1.05 (s, 3H), 0.99(d, 3H), 0.94-0.88 (m, 1H), 0.73-0.65 (m, 1H).

Preparation 202:

Ingenol-5,20-acetonide-3-(3-methyl-butenoate) (Compound 202)

Compound 202 was prepared according to Procedure a.

Starting material: 3-Methyl-butenoic acid.

Preparation 203:

Ingenol-5,20-acetonide-3-(2,3-dimethyl-butenoate) (Compound 203)

Compound 203 was prepared according to Procedure a.

Starting material: 2,3-Dimethyl-butenoic acid.

Preparation 204:

Ingenol-5,20-acetonide-3-(2-methylene-butanoate) (Compound 204)

Compound 204 was prepared according to Procedure a.

Starting material: 2-Methylene-butanoic acid.

Preparation 205:

Ingenol-5,20-acetonide-3-(2-methyl-propanoate) (Compound 205)

Compound 205 was prepared according to Procedure a.

Starting material: 2-Methyl-propanoic acid.

¹H NMR (300 MHz, CDCl₃) δ 6.05-6.04 (m, 1H), 5.78-5.76 (m, 1H), 5.54 (s,1H), 4.24-4.10 (m, 3H), 4.00 (s, 1H), 3.09 (s, 1H), 2.65-2.54 (m, 2H),2.31-2.22 (m, 1H), 1.80-1.71 (m, 4H), 1.45 (s, 3H), 1.41 (s, 3H), 1.21(d, 3H), 1.19 (d, 3H), 1.09 (s, 3H), 1.05 (s, 3H), 0.98 (d, 3H),0.93-0.87 (m, 1H), 0.73-0.65 (m, 1H).

Preparation 206:

Ingenol-5,20-acetonide-3-(3-methyl-butanoate) (Compound 206

Compound 206 was prepared according to Procedure a.

Starting material: 3-Methyl-butanoic acid.

¹H NMR (300 MHz, CDCl₃) δ 6.04-6.03 (m, 1H), 5.79-5.77 (m, 1H), 5.55 (s,1H), 4.24-4.10 (m, 3H), 4.00 (s, 1H), 3.15 (s, 1H), 2.60-2.52 (m, 1H),2.32-2.04 (m, 4H), 1.80-1.71 (m, 4H), 1.45 (s, 3H), 1.41 (s, 3H), 1.09(s, 3H), 1.05 (s, 3H), 0.99 (d, 6H), 0.98 (d, 3H), 0.94-0.87 (m, 1H),0.73-0.65 (m, 1H).

Preparation 207:

Ingenol-5,20-acetonide-3-(2(RS)-methyl-butanoate) (Compound 207a and207b)

Compound 207a and 207b were prepared according to Procedure a.

Starting material: (RS)-2-methyl-butanoic acid.

¹H NMR (300 MHz, CDCl₃) δ 6.05-6.03 (m, 1H), 5.79-5.76 (m, 1H), 5.55 (s,1H), 4.24-4.10 (m, 3H), 4.00 (s, 1H), 3.12 (s, 1H), 2.61-2.53 (m, 1H),2.48-2.40 (m, 1H), 2.31-2.22 (m, 1H), 1.80-1.66 (m, 5H), 1.56-1.47 (m,1H), 1.46 (s, 3H), 1.41 (s, 3H), 1.20-1.16 (2xd, 3H), 1.09 (s, 3H), 1.05(s, 3H), 0.99-0.87 (m, 7H), 0.73-0.65 (m, 1H).

Preparation 208:

Ingenol-5,20-acetonide-3-(3,3-dimethyl-butanoate) (Compound 208

Compound 208 was prepared according to Procedure a.

Starting material: 3,3-Dimethyl-butanoic acid.

¹H NMR (300 MHz, CDCl₃) δ 6.04-6.02 (m, 1H), 5.78-5.77 (m, 1H), 5.55 (s,1H), 4.24-4.10 (m, 3H), 4.00 (s, 1H), 3.15 (s, 1H), 2.62-2.55 (m, 1H),2.30-2.20 (m, 1H), 2.27 (s, 2H), 1.78-1.69 (m, 4H), 1.45 (s, 3H), 1.41(s, 3H), 1.10 (s, 3H), 1.06 (s, 9H), 1.05 (s, 3H), 0.97 (d, 3H),0.93-0.87 (m, 1H), 0.73-0.65 (m, 1H).

Preparation 209:

Ingenol-5,20-acetonide-3-(2-ethyl-butanoate) (Compound 209)

Compound 209 was prepared according to Procedure a.

Starting material: 2-Ethyl-butanoic acid.

¹H NMR (300 MHz, CDCl₃) δ 6.05-6.03 (m, 1H), 5.78-5.76 (m, 1H), 5.56 (s,1H), 4.25-4.10 (m, 3H), 4.01 (s, 1H), 3.16 (s, 1H), 2.64-2.54 (m, 1H),2.31-2.21 (m, 2H), 1.78-1.48 (m, 8H), 1.46 (s, 3H), 1.41 (s, 3H), 1.09(s, 3H), 1.04 (s, 3H), 0.98 (d, 3H), 0.94 (t, 3H), 0.93 (t, 3H),0.93-0.87 (m, 1H), 0.72-0.65 (m, 1H).

Preparation 210:

Ingenol-5,20-acetonide-3-(2R-methyl-butanoate) (Compound 210)

Compound 210 was prepared according to Procedure a.

Starting material: (R)-2-Methyl-butanoic acid.

¹H NMR (300 MHz, CDCl₃) δ6.05-6.03 (m, 1H), 5.79-5.76 (m, 1H), 5.55 (s,1H), 4.24-4.10 (m, 3H), 4.00 (s, 1H), 3.12 (s, 1H), 2.61-2.53 (m, 1H),2.50-2.39 (m, 1H), 2.31-2.22 (m, 1H), 1.79-1.66 (m, 5H), 1.59-1.47 (m,1H), 1.45 (s, 3H), 1.41 (s, 3H), 1.19-1.16 (d, 3H), 1.09 (s, 3H), 1.05(s, 3H), 0.99-0.87 (m, 7H), 0.73-0.65 (m, 1H).

Preparation 211:

Ingenol-5,20-acetonide-3-tiglate (Compound 211)

Compound 211 was prepared according to Procedure a.

Starting material: Tiglic acid.

¹H NMR (300 MHz, CDCl₃) δ 6.90-6.82 (m, 1H), 6.05-6.04 (m, 1H),5.78-5.76 (m, 1H), 5.62 (s, 1H), 4.24-4.11 (m, 3H), 4.01 (s, 1H), 3.17(s, 1H), 2.61-2.56 (m, 1H), 2.31-2.22 (m, 1H), 1.86-1.72 (m, 10H), 1.45(s, 3H), 1.42 (s, 3H), 1.09 (s, 3H), 1.05 (s, 3H), 1.00 (d, 3H),0.94-0.88 (m, 1H), 0.73-0.63 (m, 1H).

Preparation 212:

Ingenol-5,20-acetonide-3-(phenyl-acetate) (Compound 212)

Compound 212 was prepared according to Procedure a.

Starting material: Phenyl-acetic acid.

Preparation 213:

Ingenol-5,20-acetonide-3-(2Z-(methoxycarbonyl)-acrylate) (Compound 213)

Compound 213 was prepared according to Procedure a.

Starting material: Fumaric acid mono-methyl ester.

¹H NMR (300 MHz, CDCl₃) δ 6.93 (d, 1H), 6.85 (d, 1H), 6.09 (m, 1H),6.80-6.78 (m, 1H), 5.62 (s, 1H), 4.26-4.09 (m, 3H), 4.02 (s, 1H), 3.82(s, 3H), 3.59-3.53 (m, 1H), 2.29-2.20 (m, 1H), 2.82-2.73 (m, 4H), 1.47(s, 3H), 1.43 (s, 3H), 1.27 (bs, 1H), 1.09 (s, 3H), 1.05 (s, 3H), 1.00(d, 3H), 0.94-0.85 (m, 1H), 0.73-0.65 (m, 1H).

Preparation 214:

Ingenol-5,20-acetonide-3-(2-cyclohexylpropanoate) (Compound 214)

Compound 214 was prepared according to Procedure c.

Starting material: 2-Cyclohexylpropanoic acid.

Preparation 215:

Ingenol-5,20-acetonide-3-((2Z)-2-methoxyimino-2-phenyl-acetate)(Compound 215)

Compound 215 was prepared according to Procedure c.

Starting material: (2Z)-2-Methoxyimino-2-phenyl-acetic acid.

Preparation 216:

Ingenol-5,20-acetonide-3-((2E)-2-methoxyimino-2-phenyl-acetate)(Compound 216)

Compound 216 was prepared according to Procedure c.

Starting material: (2E)-2-Methoxyimino-2-phenyl-acetic acid.

¹H NMR (300 MHz, CDCl₃) δ 7.47-7.36 (m, 5H), 6.03-6.02 (m, 1H),5.78-5.76 (m, 1H), 5.70 (s, 1H), 4.23-4.09 (m, 3H), 4.05 (s, 3H), 4.01(s, 1H), 3.32 (s, 1H), 2.28-2.13 (m, 2H), 1.77 (d, 3H), 1.68-1.57 (m,1H), 1.45 (s, 3H), 1.41 (s, 3H), 1.10 (s, 3H), 1.05 (s, 3H), 0.92-0.86(m, 1H), 0.81 (d, 3H), 0.70-0.62 (m, 1H).

Preparation 217:

Ingenol-5,20-acetonide-3-((Z)-2-methyl-3-(p-tolyl)prop-2-enoate)(Compound 217)

Compound 217 was prepared according to Procedure c.

Starting material: (Z)-2-Methyl-3-(p-tolyl)prop-2-enoic acid.

Preparation 218:

Ingenol-5,20-acetonide-3-((E)-2-phenylbut-2-enoate) (Compound 218)

Compound 218 was prepared according to Procedure c.

Starting material: (E)-2-phenylbut-2-enoic acid.

Preparation 219:

Ingenol-5,20-acetonide-3-(2,2-diphenylacetate) (Compound 219)

Compound 219 was prepared according to Procedure c.

Starting material: 2,2-Diphenylacetic acid.

Preparation 220:

Ingenol-5,20-acetonide-3-(2-cyano-2-cyclohexylidene-acetate) (Compound220)

Compound 220 was prepared according to Procedure d, but extending thereaction time to 40 min.

Starting material: 2-cyano-2-cyclohexylidene-acetyl chloride, preparedfrom 2-cyano-2-cyclohexylidene-acetic acid by reaction with 1.25 eq.oxalyl chloride in dichloromethane and a drop of N,N-dimethylformamideat room temperature for 30 min followed by evaporation of volatiles invacuum.

Preparation 221:

Ingenol-5,20-acetonide-3-(2-(methyl(phenyl)amino)-2-oxo-acetate)(Compound 221)

Compound 221 was prepared according to Procedure c.

Starting material: 2-(methyl(phenyl)amino)-2-oxo-acetic acid.

¹H NMR (300 MHz, CDCl₃) δ 7.41-7.24 (m, 5H), 5.99-5.98 (m, 1H),5.76-5.73 (m, 1H), 5.39 (s, 1H), 4.19-4.02 (m, 3H), 3.87-3.86 (m, 1H),3.36 (s, 3H), 2.75 (s, 1H), 2.46-2.41 (m, 1H), 2.24-2.15 (m, 1H),1.79-1.67 (m, 1H), 1.51 (d, 3H), 1.36 (s, 3H), 1.32 (s, 3H), 1.09 (s,3H), 1.06 (s, 3H), 0.94 (d, 3H), 0.91-0.84 (m, 1H), 0.72-0.64 (m, 1H).

Example 101 Ingenol 3-(2-methyl-acrylate) (Compound 101)

Compound 101 was prepared according to Procedure e.

Starting material: Compound 201.

¹H NMR (300 MHz, CDCl₃) δ 6.15 (s, 1H), 6.06-6.04 (m, 2H), 5.65-5.64 (m,1H), 5.56 (s, 1H), 4.20-4.11 (m, 3H), 4.05 (s, 1H), 3.49 (bs, 1H),2.9-2.7 (bs, 2H), 2.55-2.50 (m, 1H), 2.31-2.22 (m, 1H), 1.99 (s, 3H),1.79 (d, 3H), 1.80-1.72 (m, 1H), 1.09 (s, 3H), 1.05 (s, 3H), 0.98 (d,3H), 0.98-0.91 (m, 1H), 0.74-0.65 (m, 1H).

Example 102 Ingenol 3-(3-methyl-butenoate) (Compound 102) Compound 102was prepared according to Procedure e.

Starting material: Compound 202.

¹H NMR (300 MHz, CDCl₃) δ 6.05 (d, 1H), 6.02-6.01 (m, 1H), 5.77-5.76 (m,1H), 5.51 (s, 1H), 4.15-4.09 (m, 3H), 4.03 (s, 1H), 3.48 (bs, 1H), 2.79(bs, 2H), 2.54-2.49 (m, 1H), 2.30-2.20 (m, 1H), 2.20 (d, 3H), 1.94 (d,3H), 1.88-1.72 (m, 1H), 1.78 (d, 3H), 1.09 (s, 3H), 1.05 (s, 3H),0.98-0.91 (m, 4H), 0.73-0.65 (m, 1H).

Example 103 Ingenol 3-(2,3-dimethyl-butenoate) (Compound 103)

Compound 103 was prepared according to Procedure e.

Starting material: Compound 203.

¹H NMR (300 MHz, CDCl₃) δ 6.07-6.05 (d, 1H), 6.03-6.02 (m, 1H), 5.50 (s,1H), 4.15-4.09 (m, 3H), 4.05 (s, 1H), 3.47 (bs, 1H), 2.55-2.48 (m, 1H),2.30-2.21 (m, 1H), 2.07 (m, 3H), 1.89 (m, 3H), 1.85 (s, 3H), 1.80 (m,3H), 1.99-1.55 (m, 3H), 1.09 (s, 3H), 1.05 (s, 3H), 0.96-0.88 (d, 4H),0.74-0.65 (m, 1H).

Example 104 Ingenol 3-(2-methylene-butyrate) (Compound 104)

Compound 104 was prepared according to Procedure e.

Starting material: Compound 204.

¹H NMR (300 MHz, CDCl₃) δ 6.19 (s, 1H), 6.06-6.04 (m, 2H), 5.61 (m, 1H),5.57 (s, 1H), 4.20-4.11 (m, 3H), 4.05 (s, 1H), 3.5 (bs, 1H), 2.9 (bs,2H), 2.55-2.50 (m, 1H), 2.39-2.22 (m, 3H), 1.79 (d, 3H), 1.80-1.72 (m,1H), 1.11 (t, 3H), 1.09 (s, 3H), 1.05 (s, 3H), 0.98 (d, 3H), 0.97-0.91(m, 1H), 0.74-0.65 (m, 1H).

Example 105 Ingenol 3-(2-methyl-propanoate) (Compound 105)

Compound 105 was prepared according to Procedure e.

Starting material: Compound 205.

¹H NMR (300 MHz, CDCl₃) δ 6.05-6.02 (m, 2H), 5.45 (s, 1H), 4.19-4.11 (m,3H), 4.03 (s, 1H), 3.45 (bs, 1H), 2.65 (m, 1H), 2.54-2.49 (m, 1H), 2.5(bs, 1H), 2.31-2.22 (m, 1H), 1.81-1.71 (m, 1H), 1.77 (d, 3H), 1.23 (d,3H), 1.21 (d, 3H), 1.09 (s, 3H), 1.05 (s, 3H), 0.97 (d, 3H), 0.97-0.86(m, 2H), 0.73-0.65 (m, 1H).

Example 106 Ingenol 3-(3-methyl-butanoate) (Compound 106)

Compound 106 was prepared according to Procedure e.

Starting material: Compound 206.

¹H NMR (300 MHz, CDCl₃) δ 6.05-6.02 (m, 2H), 5.43 (s, 1H), 4.19-4.11 (m,3H), 4.03 (s, 1H), 3.49 (bs, 1H), 2.6-2.4 (bs, 1H), 2.53-2.45 (m, 1H),2.31-2.23 (m, 3H), 2.17-2.04 (m, 1H), 1.81-1.71 (m, 1H), 1.78 (d, 3H),1.27 (bs, 1H), 1.09 (s, 3H), 1.05 (s, 3H), 1.00 (d, 6H), 0.97 (d, 3H),0.97-0.86 (m, 1H), 0.73-0.66 (m, 1H).

Example 107 Ingenol 3-(RS-2-methyl-butanoate) (Compound 107a andCompound 107b)

Compound 107a and 107b were prepared according to Procedure e.

Starting material: Compound 207a and 207b.

¹H NMR (300 MHz, CDCl₃) δ 6.06-6.02 (m, 2H), 5.46 (s, 1H), 4.19-4.11 (m,3H), 4.03 (s, 1H), 3.45 (bs, 1H), 2.6 (bs, 1H), 2.54-2.41 (m, 2H),2.31-2.22 (m, 1H), 1.78 (d, 3H), 1.80-1.64 (m, 2H), 1.57-1.47 (m, 1H),1.28 (bs, 1H), 1.21-1.18 (m, 3H), 1.09 (s, 3H), 1.05 (s, 3H), 0.98-0.85(m, 7H), 0.73-0.65 (m, 1H).

Example 108 Ingenol 3-(3,3-dimethyl-butanoate) (Compound 108)

Compound 108 was prepared according to Procedure e.

Starting material: Compound 208.

¹H NMR (300 MHz, CDCl₃) δ 6.06-6.02 (m, 2H), 5.47 (s, 1H), 4.19-4.11 (m,3H), 4.03 (s, 1H), 3.51 (s, 1H), 2.6-2.4 (bs, 1H), 2.54-2.49 (m, 1H),2.30 (s, 2H), 2.30-2.21 (m, 1H), 1.78 (d, 3H), 1.77-1.70 (m, 1H), 1.27(bs, 1H), 1.09 (s, 3H), 1.06 (s, 9H), 1.05 (s, 3H), 0.96 (d, 3H),0.98-0.88 (m, 1H), 0.73-0.65 (m, 1H).

Example 109 Ingenol 3-(2-ethyl-butanoate) (Compound 109)

Compound 109 was prepared according to Procedure e.

Starting material: Compound 209.

¹H NMR (300 MHz, CDCl₃) δ 6.06-6.03 (m, 2H), 5.43 (s, 1H), 4.19-4.11 (m,3H), 4.04 (s, 1H), 3.50 (s, 1H), 2.6-2.4 (bs, 1H), 2.54-2.49 (m, 1H),2.34-2.22 (m, 2H), 1.78 (d, 3H), 1.79-1.51 (m, 5H), 1.27 (bs, 1H), 1.09(s, 3H), 1.05 (s, 3H), 0.98-0.89 (m, 10H), 0.73-0.65 (m, 1H).

Example 110 Ingenol 3-(2R-methyl-butanoate) (Compound 110)

Compound 110 was prepared according to Procedure e.

Starting material: Compound 210.

¹H NMR (300 MHz, CDCl₃) δ 6.06-6.02 (m, 2H), 5.46 (s, 1H), 4.19-4.11 (m,3H), 4.03 (s, 1H), 3.46 (bs, 1H), 2.6-2.4 (bs, 1H), 2.54-2.44 (m, 2H),2.31-2.22 (m, 1H), 1.78 (d, 3H), 1.80-1.66 (m, 2H), 1.57-1.48 (m, 1H),1.27 (bs, 1H), 1.19 (d, 3H), 1.09 (s, 3H), 1.05 (s, 3H), 0.98-0.86 (m,7H), 0.73-0.65 (m, 1H).

Example 111 Ingenol 3-tiglate (Compound 111)

Compound 111 was prepared according to Procedure e.

Starting material: Compound 211.

¹H NMR (300 MHz, CDCl₃) δ 6.95-6.88 (m, 1H), 6.05-6.03 (m, 2H), 5.51 (s,1H), 4.14-4.11 (m, 3H), 4.05 (s, 1H), 3.47 (bs, 1H), 2.77 (bs, 2H),2.54-2.49 (m, 1H), 2.30-2.21 (m, 1H), 1.87-1.72 (m, 10H), 1.09 (s, 3H),1.05 (s, 3H), 0.98 (d, 3H), 0.98-0.91 (m, 1H), 0.73-0.65 (m, 1H).

Example 112 Ingenol 3-(phenyl-acetate) (Compound 112)

Compound 112 was prepared according to Procedure e.

Starting material: Compound 212.

¹H NMR (300 MHz, CDCl₃) δ 7.36-7.25 (m, 5H), 6.00-5.98 (m, 2H), 5.53 (s,1H), 4.16-4.00 (m, 3H), 3.94 (s, 1H), 3.72 (s, 2H), 3.14 (bs, 1H),2.3-1.9 (bs, 1H), 2.23-2.18 (m, 1H), 2.09-1.99 (m, 1H), 1.74 (d, 3H),1.69-1.59 (m, 1H), 1.27 (bs, 1H), 1.05 (s, 6H), 0.93-0.85 (m, 4H),0.70-0.62 (m, 1H).

Example 113 Ingenol 3-(2Z-(methoxycarbonyl))-acrylate (Compound 113)

Compound 113 was prepared according to Procedure e.

Starting material: Compound 213.

¹H NMR (300 MHz, CDCl₃) δ 6.95 (d, 1H), 6.88 (d, 1H), 6.09 (m, 1H), 6.05(d, 1H), 5.65 (s, 1H), 4.54 (d, 1H), 4.20-4.13 (m, 3H), 4.05 (d, 1H),3.83 (s, 3H), 3.57 (s, 1H), 2.54-2.43 (m, 2H), 2.31-2.22 (m, 1H),1.81-1.72 (m, 4H), 1.07 (s, 3H), 1.05 (s, 3H), 0.97 (d, 3H), 0.95-0.89(m, 1H), 0.74-0.65 (m, 1H).

Example 114 Ingenol 3-(2-cyclohexylpropanoate) (Compound 114)

Compound 114 was prepared according to Procedure e.

Starting material: Compound 214.

¹H NMR (300 MHz, CDCl₃) δ 6.06-6.02 (m, 2H), 5.46 (d, 1H), 3.32 (t, 1H),4.19-4.11 (m, 3H), 4.04-4.03 (m, 1H), 3,52 (s, 1H), 2.55-2.47 (m, 1H),2.39-2.23 (m, 2H), 1.80-1.60 (m, 10H), 1.30-0.86 (m, 19H), 0.73-0.65 (m,1H).

Example 115 Ingenol 3-((2Z)-2-methoxyimino-2-phenyl-acetate) (Compound115)

Compound 115 was prepared according to Procedure e.

Starting material: Compound 215.

¹H NMR (300 MHz, CDCl₃) δ 7.70-7.66 (m, 2H), 7.45-7.38 (m, 3H),6.12-6.11 (m, 1H), 6.09 (s, 1H), 6.07-6.05 (m, 1H), 4.22-4.12 (m, 4H),4.07 (s, 3H), 4.00 (d, 1H), 3.58 (d, 1H), 2.41-2.36 (m, 1H), 2.29-2.18(m, 2H), 1.83 (d, 3H), 1.80-1.73 (m, 1H), 1.12 (s, 3H), 1.07 (s, 3H),0.96-0.86 (m, 4H), 0.75-0.67 (m, 1H).

Example 116 Ingenol 3-((2E)-2-methoxyimino-2-phenyl-acetate) (Compound116)

Compound 116 was prepared according to Procedure e.

Starting material: Compound 216.

¹H NMR (300 MHz, CDCl₃) δ 7.70-7.66 (m, 2H), 7.45-7.38 (m, 3H),6.12-6.05 (m, 3H), 4.22-4.09 (m, 4H), 4.07 (s, 3H), 4.00 (d, 1H), 3.60(d, 1H), 2.41-2.36 (m, 1H), 2.29-2.20 (m, 2H), 1.83 (d, 3H), 1.80-1.73(m, 1H), 1.12 (s, 3H), 1.07 (s, 3H), 0.99-0.92 (m, 4H), 0.75-0.67 (m,1H).

Example 117 Ingenol 3-((Z)-2-methyl-3-(p-tolyl)prop-2-enoate) (Compound117)

Compound 117 was prepared according to Procedure e.

Starting material: Compound 217.

¹H NMR (300 MHz, CDCl₃) δ 7.70 (s, 1H), 7.32 (d, 2H), 7.21 (d, 2H),6.07-6.05 (m, 2H), 5.60 (s, 1H), 4.44 (d, 1H), 4.17-4.08 (m, 4H), 3.55(s, 1H), 2.61-2.49 (m, 2H), 2.38 (s, 3H), 2.31-2.23 (m, 1H), 2.15 (d,3H), 1.83 (d, 3H), 1.80-1.73 (m, 1H), 1.09 (s, 3H), 1.04 (s, 3H), 1.01(d, 3H), 0.98-0.92 (m, 1H), 0.74-0.66 (m, 1H).

Example 118 Ingenol 3-((E)-2-phenylbut-2-enoate) (Compound 118)

Compound 118 was prepared according to Procedure e.

Starting material: Compound 218.

¹H NMR (300 MHz, CDCl₃) δ 7.39-7.30 (m, 3H), 7.24 (q, 1H), 7.20-7.15 (m,2H), 5.99-5.96 (m, 1H), 5.95-5.93 (m, 1H), 5.57 (s, 1H), 4.16-3.93 (m,4H), 3.80 (d, 1H), 3.19 (s, 1H), 2.34 (t, 1H), 1.95-1.83 (m, 2H), 1.78(s, 3H), 1.77 (d, 3H), 1.57-1.46 (m, 1H), 1.05 (s, 3H), 1.04 (s, 3H),0.93-0.85 (m, 1H), 0.76 (d, 3H), 0.65-0.58 (m, 1H).

Example 119 Ingenol 3-(2,2-diphenylacetate) (Compound 119)

Compound 119 was prepared according to Procedure e.

Starting material: Compound 219.

¹H NMR (300 MHz, CDCl₃) δ 7.35-7.25 (m, 10H), 6.01-5.96 (m, 2H), 5.62(s, 1H), 5.12 (s, 1H), 4.13-3.93 (m, 5H), 3.14 (s, 1H), 2.34 (t, 1H),2.12-2.05 (m, 1H), 2.04-1.93 (m, 1H), 1.72 (d, 3H), 1.61-1.52 (m, 1H),1.05 (s, 3H), 1.04 (s, 3H), 0.91-0.83 (m, 4H), 0.67-0.59 (m, 1H).

Example 120 Ingenol 3-(2-cyano-2-cyclohexylidene-acetate) (Compound 120)

Compound 120 was prepared according to Procedure e.

Starting material: Compound 220.

¹H NMR (300 MHz, CDCl₃) δ 6.12-6.10 (m, 1H), 6.08-6.06 (m, 1H), 5.67 (s,1H), 4.20-4.02 (m, 5H), 3.56 (s, 1H), 3.02 (t, 2H), 2.71-2.63 (m, 3H),2.32-2.23 (m, 1H), 2.13-2.08 (m, 1H), 1.85-1.65 (m, 10H), 1.09 (s, 3H),1.06 (s, 3H), 1.00 (d, 3H), 0.96-0.88 (m, 1H), 0.75-0.67 (m, 1H).

Example 121 Ingenol 3-(2-(methyl(phenyl)amino)-2-oxo-acetate) (Compound121)

Compound 121 was prepared according to Procedure e.

Starting material: Compound 221.

¹H NMR (300 MHz, CDCl₃) δ 7.43-7.33 (m, 3H), 7.28-7.24 (m, 2H),6.00-5.98 (m, 2H), 5.52 (s, 1H), 4.16-4.02 (m, 3H), 3.84 (s, 2H), 3.37(s, 3H), 3.29 (s, 1H), 2.38-2.32 (m, 2H), 2.24-2.14 (m, 1H), 1.81-1.69(m, 1H), 1.50 (d, 3H), 1.08 (s, 3H), 1.06 (s, 3H), 0.96 (d, 3H),0.93-0.87 (m, 1H), 0.73-0.65 (m, 1H).

Example 1

Neutrophil Oxidative Burst Assay:

PMN's (polymorphonuclear leukocytes) were isolated and purified fromfresh buffy coats by sequential sedimentation, density centrifugationand lysis of contaminating erythrocytes. Buffy coats were incubated with2% methocel for 30-45 min to differentially sediment red blood cells.The leukocyte-rich supernatant was transferred to lymphoprep tubes toremove mononuclear cells by density centrifugation (400×g, 30 min). Thepellet was resuspended and any remaining erythrocytes lysed using 0.2%NaCl for 30 sec before restoring isotonicity by the addition of 1.2%NaCl. This step was repeated until the cell pellet appears relativelyfree of red blood cells. Cells were resuspended in DPBS (Dulbecco'sPhosphate Buffered Saline) (w.o. Ca²⁺, Mg²⁺) and the concentrationadjusted to 1.4×10⁶ cells/ml in HBSS (Hanks Balanced Salt solution) (wCa²⁺, Mg²⁺) containing 0.1% BSA (Bovine Serum Albumin) and 5 mM glucosejust prior to assay initiation. Titrated reference and test compoundswere pre-mixed with HE (Hydroethidine) (10 μM final assay concentration)before addition to 96-well plates containing 2.5×10⁵ cells. Following 40min incubation at RT, changes in the respiratory burst was estimated bymeasuring fluorescence at 579 nm (excitation: 485 nm) using an Envisionplate reader.

Test compound titration curves were fitted to a four-parameter sigmoidalcurve after normalizing the effect of the test compound to the effect ofthe positive control (5×10⁻⁷ M PEP0005). Rel EC₅₀ denotes theconcentration of test compound producing an effect that is midwaybetween the fitted top and bottom. Abs EC₅₀ is the concentration of testcompound that provokes a response corresponding to 50% of the maximaleffect associated with the positive control (5×10⁻⁷ M PEP0005).

Example 2

HeKa Cytokine Release (IL-8) Assay:

Primary human epidermal keratinocytes, HeKa, were seeded (10.000cells/well) in 96-well plates the day before the assay. Test compoundswere diluted in DMSO (dimethyl sulfoxide) and further diluted in assaymedium and pipetted into wells of 96 well-plates containing HeKa cells.The plates were incubated for 6 h at 37° C. in humidified air with 5%CO₂. Plates were centrifuged briefly to spin down cells at 4° C., thesupernatant was removed and analysed by Meso Scale Discovery (MSD)4-spot cytokine assay (Pro-inflammatory II Ultra Sensitive kit, MSD, MD,USA). The MSD assay employs a sandwich immunoassay format where captureantibodies are coated in a patterned array on the bottom of the wells ofa 4-Spot-Multi-MSD plate. Standard samples were incubated in theMULTI-SPOT plates as well, and the cytokine (IL-8) binds to itscorresponding capture antibody spot. The cytokine level was quantitatedon a SECTOR™ Imager using a cytokine-specific Detection Antibodylabelled with MSD SULFO-TAG™ reagent.

Test compound titration curves were fitted to a four-parameter sigmoidalcurve after normalizing the effect of the test compound to the effect ofthe positive control (1.5×10⁻⁷ M PEP0005). Rel EC₅₀ denotes theconcentration of test compound producing an effect that is midwaybetween the fitted top and bottom. Abs EC₅₀ is the concentration of testcompound that provokes a response corresponding to 50% of the maximaleffect associated with the positive control (1.5×10⁻⁷ M PEP0005).

Example 3

Necrosis Assay

HeLa cells (ATCC CCL-002) were grown in minimal essential medium(Invitrogen catalog no. 42360) containing 10% fetal bovine serum, 100IU/ml penicillin and 100 μg/ml streptomycin. 4,000-6,000 cells wereseeded into 96-well black ViewPlates-plates, clear bottom, (PerkinElmer) in 100 μl medium and incubated overnight. Compounds weredissolved and pre-diluted in DMSO in 96-well polypropylene plates(Greiner) in a concentration range of 15 μM to 600 μM. At the time ofthe experiment cell plates were placed on heating blocks at 37° C.,medium was removed and 40 μl fresh, pre-warmed medium was added perwell. Cells were incubated for 15 min before addition of compounds. Inparallel, 3 μl of compounds were diluted with 197 μl growth medium on aTecan freedom-EVO pipetting station using 250 μl/s pipetting speed, inorder to ensure effective mixing of the highly concentrated compoundsolutions with the aqueous phase. These pre-dilution plates were thenequilibrated on heating blocks at 37° C. for 10 min. 80 μl pre-dilutedcompound were transferred manually to the corresponding wells containingHeLa cells yielding compound concentrations of 10 μM to 400 μM. Controlconditions were 1% DMSO in growth medium (100% viability) and 400 μMingenol mebutate in growth medium (0% viability). Plates were incubatedon the heating blocks at 37° C. for 30 min. At the end of the incubation10 μl PrestoBlue reagent (Invitrogen) were added to each well, plateswere sealed with black seal, followed by incubation at 37° C. for 10 minwith gentle shaking (150 rpm). Subsequently, plates were placed at roomtemperature for 20-30 min. Plates were read immediately after on anEnvision Fluorescence reader (Perkin Elmer) with excitation at 535 nmand emission at 630 nm. Test compound titration curves were fitted to afour-parameter sigmoidal curve after normalizing the effect of the testcompound to the effect of the positive control (4 10⁻⁴ M PEP0005/ingenolmebutate). AbsEC₅₀ denotes the concentration of test compound producing50% effect.

Compounds of the present invention were tested in the neutrophiloxidative burst assay according to the description in example 1, in theHeKa cytokine release assay according to the description in example 2and in the necrosis assay according to the description in example 3.

Neutrophil oxidative burst Rel EC₅₀ ranges

-   * indicates that Rel EC₅₀ values are ≧100 nM-   ** indicates that Rel EC₅₀ values are ≧20 nM and <100 nM-   *** indicates that Rel EC₅₀values are <20 nM

HeKa cytokine release (IL-8) Rel EC₅₀ ranges p0 * indicates that RelEC₅₀ values are ≧100 nM

-   ** indicates that Rel EC₅₀ values are ≧20 nM and <100 nM-   *** indicates that Rel EC₅₀values are <20 nM

HeLa Necrosis EC₅₀ ranges

-   * indicates that EC₅₀ values are ≦350 μM-   ** indicates that EC₅₀ values are ≧150 μM and <350 μM-   *** indicates that EC₅₀values are <150 μM

HeKa Neutrophil cytokine oxidative release HeLa burst (IL-8) NecrosisRel EC₅₀ Rel EC₅₀ EC₅₀ Compound name and number range range rangeIngenol 3-(2-methyl-acrylate) *** * ** (Compound 101) Ingenol3-(3-methyl-butenoate) *** ** ** (Compound 102) Ingenol3-(2,3-dimethyl-butenoate) *** *** ** (Compound 103) Ingenol3-(2-methylene-butyrate) *** ** ** (Compound 104) Ingenol3-(2-methyl-propanoate) *** ** — (Compound 105) Ingenol3-(3-methyl-butanoate) *** ** — (Compound 106) Ingenol3-(RS-2-methyl-butanoate) *** ** — (Compound 107a and Compound 107b)Ingenol 3-(3,3-dimethyl-butanoate) *** ** ** (Compound 108) Ingenol3-(2-ethyl-butanoate) *** *** ** (Compound 109) Ingenol3-(2R-methyl-butanoate) *** ** — (Compound 110) Ingenol 3-tiglate(Compound 111) *** ** * Ingenol 3-(phenyl-acetate) *** * ** (Compound112) Ingenol 3-(2Z-(methoxycarbonyl)- * — — acrylate (Compound 113)Ingenol 3-(2-cyclohexylpropanoate) *** *** ** (Compound 114) Ingenol3-((2Z)-2-methoxyimino-2- ** *** *** phenyl-acetate) (Compound 115)Ingenol 3-((2E)-2-methoxyimino-2- ** ** — phenyl-acetate) (Compound 116)Ingenol 3-((Z)-2-methyl-3-(p- *** *** *** tolyl)prop-2-enoate) (Compound117) Ingenol 3-((E)-2-phenylbut-2- *** *** *** enoate) (Compound 118)Ingenol 3-(2,2-diphenylacetate) *** *** *** (Compound 119) Ingenol3-(2-cyano-2- *** * — cyclohexylidene-acetate) (Compound 120) Ingenol3-(2- ** * * (methyl(phenyl)amino)-2-oxo- acetate) (Compound 121)PEP005, Ingenol-3-angelate *** *** **

1. A compound of the general formula I

wherein R is (C₁-C₇)alkyl, (C₂-C₇)alkenyl or (C₂-C₇)alkynyl; wherein Ris substituted one or more times with substituents independentlyselected from R1; wherein R1 represents (a) Fluoro, cyano or hydroxy; or(b) (C₁-C₄)alkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl,heterocycloalkyl, heterocycloalkenyl or (C₃-C₆)cycloalkylidene each ofwhich may optionally be substituted by one or more substituentsindependently selected from R2, wherein R2 represents halogen, cyano,hydroxy, (C₁-C₄)alkyl, (C₂-C₄)alkenyl, halo(C₁-C₄)alkyl, —NRaCORb,—COORc, —OCORa, —CONRaRb, —OCONRaRb, —NRaCOORb, —NRaCONRaRb, —NRaSO2Rb,—NRaSO2NRaRb, —SO2NRaRb, —SO2Ra, —S(O)Ra, —ORa, —SRa, ═N—ORa, ═O; or (c)—NRaCORb, —COORc, —OCORa, —CONRaRb, —OCONRaRb, —NRaCOORb, —NRaCONRaRb,—NRaSO2Rb, —NRaSO2NRaRb, —SO2NRaRb, —SO2Ra, —S(O)Ra, —ORa, —SRa, ═N—ORa,—NRaRb or ═O; and wherein Ra and Rb independently represents hydrogen,(C₁-C₄)alkyl, halo(C₁-C₄)alkyl, (C₁-C₄)alkoxyl(C₁-C₄)alkyl,hydroxy(C₁-C₄)alkyl, cyano(C₁-C₄)alkyl or aryl; and wherein Rcrepresents (C₁-C₄)alkyl, halo(C₁-C₄)alkyl, (C₁ -C₄)alkoxyl(C₁ -C₄)alkyl,hydroxy(C₁-C₄)alkyl or cyano(C₁-C₄)alkyl, and pharmaceuticallyacceptable salts, hydrates and solvates thereof; with the proviso whenR1 represents alkyl or alkenyl the combined length of R and R1 does notexceed that of a chain of 7 carbon atoms, and with the proviso that Rand R1 in combination may not be a straight unbranched or unsubstitutedalkyl chain or a straight unbranched or unsubstituted alkenyl chain, andwith the proviso that R, or R and R1 in combination is nottrans-2-buten-2-yl.
 2. A compound according to claim 1, wherein R issubstituted by R1 in the alpha- or beta-position relative to thecarbonyl-group.
 3. A compound according to claim 1, wherein R issubstituted by R1 in the alpha-position relative to the carbonyl-group.4. A compound according to claim 1, wherein R is C₁-C₇-alkyl.
 5. Acompound according to claim 4, wherein R is ethyl, propyl, tent-butyl,isopropyl, 2-butyl,3-pentyl or isobutyl.
 6. A compound according toclaim 1, wherein R is C₂-C₇-alkenyl.
 7. A compound according to claim 6wherein R is propenyl or ethenyl.
 8. A compound according to claim 1,wherein R1 is C₁-C₄-alkyl, aryl, cycloalkyl, cycloalkylidene, ═N—ORa,═O, —NRaRb, —COORc or cyano.
 9. A compound according to claim 1, whereinR1 is ethyl, methyl, phenyl, cyclohexyl, ═N—OCH3, —N(CH₃)(C₆H₅), —COOCH₃or cyclohexylidene.
 10. A compound according to claim 1, wherein R1 ismethyl.
 11. A compound according to claim 1, wherein R2 is methyl.
 12. Acompound according to claim 1, said compound being: Ingenol3-(2-methyl-acrylate), Ingenol 3-(3-methyl-butenoate), Ingenol 3-(2,3-dimethyl-butenoate), Ingenol 3-(2-methylene-butyrate), Ingenol3-(2-methyl-propanoate), Ingenol 3-(3-methyl-butanoate), Ingenol3-(2RS-methyl-butanoate), Ingenol 3-(3,3-dimethyl-butanoate), Ingenol3-(2-ethyl-butanoate), Ingenol 3-(2R-methyl-butanoate), Ingenol3-tiglate, Ingenol 3-(phenyl-acetate), Ingenol3-(2Z-(methoxycarbonyl)-acrylate), Ingenol 3-(2-cyclohexylpropanoate),Ingenol 3-((2Z)-2-methoxyimino-2-phenyl-acetate), Ingenol3-((2E)-2-methoxyimino-2-phenyl-acetate), Ingenol3-((Z)-2-methyl-3-(p-tolyl)prop-2-enoate), Ingenol3-((E)-2-phenylbut-2-enoate), Ingenol 3-(2,2-diphenylacetate), Ingenol3-(2-cyano-2-cyclohexylidene-acetate) or Ingenol3-(2-(methyl(phenyl)amino)-2-oxo-acetate). and pharmaceuticallyacceptable salts thereof.
 13. A compound according to claim 1, for useas a medicament in therapy.
 14. A compound according to claim 1 for usein the treatment, prevention, amelioration or prophylaxis ofphysiological disorders or diseases associated with hyperplasia orneoplasia.
 15. A compound according to claim 14 wherein the disorder ordisease is selected from cutaneous warts, genitial warts, actinickeratosis, squamous cell carcinoma (SCC), basal cell carcinoma (BCC),lentigo maligna, cervical intraepithelial neoplasia, analintraepithelial neoplasia or vulva intraepithelial neoplasia.
 16. Use ofa compound according to claim 1, for the manufacture of a medicament forthe treatment, amelioration or prophylaxis of physiological disorders ordiseases associated with hyperplasia or neoplasia.
 17. Use according toclaim 16 wherein the disorder or disease is selected from cutaneouswarts, genitial warts, squamous cell carcinoma (SCC), basal cellcarcinoma (BCC), lentigo maligna, cervical intraepithelial neoplasia,anal intraepithelial neoplasia or vulva intraepithelial neoplasia,neoplasia.
 18. A method of preventing, treating, amelioration orprophylaxis of physiological disorders or diseases associated withhyperplasia or neoplasia by administration to a subject in need thereofa compound according to claim
 1. 19. The method according to claim 18wherein the disorder or disease is selected from cutaneous warts,genitial warts, actinic keratosis, squamous cell carcinoma (SCC), basalcell carcinoma (BCC), lentigo maligna, cervical intraepithelialneoplasia, anal intraepithelial neoplasia or vulva intraepithelialneoplasia.
 20. A compound according to claim 1 for use in the treatmentor amelioration of cosmetic indications.
 21. A compound according toclaim 20, wherein the cosmetic indication is selected from photodamagedskin or seborrheic keratosis.
 22. Use of compound according to claim 1for the manufacture of a medicament for the treatment or amelioration ofcosmetic indications.
 23. The use according to claim 22, wherein thecosmetic indication is selected from photodamaged skin or seborrheickeratosis.
 24. A method of treatment or amelioration of cosmeticindications by administration to a subject in need thereof a compoundaccording to claim
 1. 25. The method according to claim 24 wherein thecosmetic indication is selected from photodamaged skin or seborrheickeratosis.
 26. Use of a compound according to claim 1 in the manufactureof a pharmaceutical composition for the treatment or amelioration of adisease, disorder or condition responsive to stimulation of neutrophiloxidative burst.
 27. Use of a compound according to claim 1 in themanufacture of a pharmaceutical composition for the treatment oramelioration of a disease, disorder or condition responsive tostimulation of keratinocyte IL-8 release.
 28. Use of a compoundaccording to claim 1 in the manufacture of a pharmaceutical compositionfor the treatment or amelioration of a disease, disorder or conditionresponsive to induction of necrosis.
 29. A method of preventing,treating, amelioration or prophylaxis of physiological disorders ordiseases responsive to stimulation of neutrophil oxidative burst byadministration to a subject in need thereof a compound according toclaim
 1. 30. A method of preventing, treating, amelioration orprophylaxis of physiological disorders or diseases responsive tostimulation of of keratinocyte IL-8 release by administration to asubject in need thereof a compound according to claim
 1. 31. A method ofpreventing, treating, amelioration or prophylaxis of physiologicaldisorders or diseases responsive to responsive to induction of necrosisby administration to a subject in need thereof a compound according toclaim
 1. 32. A compound according to claim 1 for use in the treatment oramelioration of a disease, disorder or condition responsive tostimulation of neutrophil oxidative burst.
 33. A compound according toclaim 1 for use in the treatment or amelioration of a disease, disorderor condition responsive to stimulation of keratinocyte IL-8 release. 34.A compound according to claim 1 for use in the treatment or ameliorationof a disease, disorder or condition responsive to induction of necrosis.35. A pharmaceutical composition comprising a compound according toclaim 1 or a pharmaceutically acceptable stereoisomer or salt thereoftogether with a pharmaceutically acceptable vehicle or excipient.
 36. Apharmaceutical composition according to claim 35, wherein thecomposition is suitable for topical administration.
 37. A pharmaceuticalcomposition comprising a compound according to claim 1 or apharmaceutically acceptable stereoisomer or salt thereof in combinationwith one or more other therapeutically active agents.